Microfluidic Devices and Methods for Use Thereof in Multicellular Assays of Secretion

ABSTRACT

Methods and devices are provided herein for identifying a cell population comprising an effector cell that exerts an extracellular effect. In one embodiment the method comprises retaining in a microreactor a cell population comprising one or more effector cells, wherein the contents of the microreactor further comprise a readout particle population comprising one or more readout particles, incubating the cell population and the readout particle population within the microreactor, assaying the cell population for the presence of the extracellular effect, wherein the readout particle population or subpopulation thereof provides a direct or indirect readout of the extracellular effect, and determining, based on the results of the assaying step, whether one or more effector cells within the cell population exerts the extracellular effect on the readout particle. If an extracellular effect is measured, the cell population is recovered for further analysis to determine the cell or cells responsible for the effect.

CROSS REFERENCE TO RELATED APPLICATION

This is a divisional of U.S. application Ser. No. 14/773,244, filed Sep.4, 2015, which is the national stage entry of PCT/CA2014/000304, filedMar. 28, 2014, which claims priority from U.S. Provisional ApplicationSer. No. 61/806,329, filed Mar. 28, 2013, the disclosure of each ofwhich is incorporated by reference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

The cell is the fundamental unit of life and no two cells are identical.For example, differences in genotype, phenotype and/or morphologicalproperty can contribute to cellular heterogeneity. Indeed, “seeminglyidentical” clonal populations of cells have been shown to displayphenotypic differences among cells within the population. Cellulardifferences exist across all levels of life, ranging from bacterialcells to partially differentiated cells (for example, adult stem andprogenitor cells) to highly differentiated mammalian cells (for example,immune cells). Differences in cellular state, function and responses canarise from a variety of mechanisms including different histories,different differentiation states, epigenetic variations, cell cycleeffects, stochastic variations, differences in genomic sequence, geneexpression, protein expression and differing cell interaction effects.

Conventional bulk cellular analyses, including measurements of expressedproteins or RNA, are performed by averaging very large numbers of cells,typically greater than 1000 cells per individual assay). This averagingof a cellular population masks the heterogeneity that exists within acell population and obscures the underlying biological features of theindividual cells within the population. There are many examples wheresuch averaged measurements are inadequate. For example, measuring acellular process in a cell population may be complicated by theresponses of individual cells, which may be asynchronous, thus blurringthe dynamics of the process. For example, the presence of dominant, yetphenotypically distinct subpopulations of cells can result in apopulation measurement that poorly reflects the internal states of themajority of cells in the population. See, e.g., Altschuler and Wu.(2010). Cell 141, pp. 559-563.

Existing methods for isolating populations of unique cell types areoften limited in the purity of the population that is achievable. Forexample, enriched populations of primary multipotent stem cells rarelyachieve better than 50% functional purity and are often well below 10%pure, so that the molecular signatures of these cells are obscured bylarge, and often overwhelming contamination from other cell types. Manycell types interact with each other, both through direct contact andthrough secreted factors, to promote survival, death, differentiation orsome other function, and these interactions are difficult to isolate andstudy in a mixture comprising a large number of cells. Additionally,cells may have differences in their genomic sequences and/or cellularstate that result in different levels or types of expressed mRNA orproteins. If analyzed in a bulk population, the particular cell with aunique cellular state or having the expressed mRNA or protein ofinterest, although of high value for industrial purposes, is verydifficult or impossible to isolate from the population.

To overcome the deficiencies of bulk population cell analysis, singlecell assay platforms have been developed. For example, microfluidicdevices have been used to study single cells in the past (Lecault et al.(2012). Curr. Opin. Chem. Biol. 16, pp. 381-390). Ma et al. (Nat Med,17, pp. 738-743 (2011)) applied a single cell barcode chip tosimultaneously measure multiple cytokines (e.g., IL-10, TNF-β, IFN-γ)from human macrophages and cytotoxic T lymphocytes (CTLs) obtained fromboth healthy donors and a metastatic melanoma patient. Microfabricatedchamber arrays have also been used to screen and select B cellssecreting antigen-specific antibodies from both immunized humans andmice (Story et al. (2009). Proc. Natl. Acad. Sci. U.S.A. 105, pp.17902-17907; Jin et al. (2009). Nat. Med. 15, pp. 1088-1092). In thisapproach, single B cells were arrayed on a surface containing tens ofthousands of microfabricated wells (˜10-100 μm deep), where the wellsurfaces were functionalized with capture antibodies. After incubationof cells on the well surfaces for less than 3 hours, the surfaces werewashed with fluorescently labeled antigen and scanned in order toidentify antigen-specific B cells. These cells were then manuallyrecovered from the arrays by a microcapillary in order to amplify,sequence, and clone the antibody-encoding genes from these cells.

Two-phase microfluidic devices have also been applied to the analysis ofsecreted proteins from single immune cells by encapsulating them insub-nanoliter aqueous droplets separated by a stream of oil (Konry etal. (2011). Biosens. Bioelectron. 26, pp. 2702-2710). These droplets canbe analyzed in a flow-through format similar to FACS, and thus providean opportunity for ultra-high throughput detection of secreted proteinsfrom single cells. Water-in-oil emulsions have also been used to studycellular paracrine signaling by co-encapsulating cells inmicrofluidic-generated agarose beads (Tumarkin et al. (2011). Integr.Biol. 3, pp. 653-662). Microfluidic droplet generation also has beenused for drug screening and development by enabling viability analysisof encapsulated single cells exposed to different compositions (Brouzeset al. (2009). Proc. Natl. Acad. Sci. U.S.A. 106, pp. 14195-14200).

Antibodies are molecules naturally produced by the immune system ofhumans or animals to fight off infection and disease. This is achievedby the unique ability of the immune system to generate an immensediversity of antibodies, each with the ability to recognize and bind aspecific target (e.g., protein, virus, bacteria). This unmatchedspecificity is also what makes antibodies extremely potent and lowside-effect drugs with clinically approved therapies for a wide array ofconditions including cancer, autoimmune disorders, inflammation,neurology, and infection. In comparison to conventional small moleculedrugs, antibodies offer several advantages including superiorpharmacokinetics, fewer side effects, improved tolerability, and muchhigher success rates in clinical trials (27% vs. 7% for smallmolecules). (Reichert (2009). Mabs 1, pp. 387-389.) It is for thisreason that antibodies are also by far the fastest growing class ofdrugs, with a total global market that was $50B in 2012 and that isgrowing at a rate of 9% per year. (Nelson et al. (2010). Nat. Rev. DrugDisc. 9(10), pp. 767-774.)

The discovery of antibodies with optimal therapeutic properties, and inparticular antibodies that target surface receptors, remains a seriousbottleneck in drug development. In response to immunization, an animalcan make millions of different monoclonal antibodies (mAbs). Each mAb isproduced by a single cell called an antibody-secreting cell (ASC), andeach ASC makes only one type of mAb. Accordingly, antibody analysis, forexample, for drug discovery purposes lends itself to single cellanalyses. However, even if an ASC is analyzed individually, and notwithin a bulk population of cells, because a single ASC generates only aminute amount of antibody, when analyzed in the volume of conventionalassay formats, the antibody is too dilute, making it completelyundetectable. Accordingly, new methods for studying individual ASCs andtheir secreted antibodies are needed. The present invention addressesthis and other needs.

SUMMARY OF THE INVENTION

The present invention provides a microfluidic platform for the analysisof an extracellular effect attributable to single effector cell. Theeffector cell, in one embodiment, is a cell that secretes a biologicalfactor, for example, an antibody (an ASC). In a further embodiment,microfluidic analysis of the effector cell is an extracellular effectassay carried out on a cell population comprising the single effectorcell.

In one aspect, a method of identifying a cell population comprising aneffector cell having an extracellular effect is provided. In oneembodiment, the method comprises retaining in a microreactor a cellpopulation comprising one or more effector cells, wherein the contentsof the microreactor further comprise a readout particle populationcomprising one or more readout particles, incubating the cell populationand the one or more readout particles within the microreactor, assayingthe cell population for the presence of the extracellular effect,wherein the readout particle population or subpopulation thereofprovides a direct or indirect readout of the extracellular effect, anddetermining, based on the results of the assaying step, whether one ormore effector cells within the cell population exhibits theextracellular effect. In a further embodiment, the microreactor is amicrofluidic chamber. In even a further embodiment, the microfluidicchamber is part of a microfluidic structure that includes membranevalves.

In this aspect, the effector cell is a cell that secretes a biologicalfactor, e.g., an antibody. It is not necessary that the specificeffector cell or effector cells, having the particular extracellulareffect be initially identified so long as the presence of theextracellular effect is detected within a particular microreactor. Thatis, some or all of the cells in the microreactor where the effect ismeasured can be recovered if desired for further characterization toidentify the specific cells providing the extracellular effect.

In one embodiment, if it is determined that a cell population comprisingone or more effector cells exhibit the extracellular effect, the cellpopulation or portion thereof is recovered to obtain a recovered cellpopulation. Recovery, in one embodiment, comprises piercing themicrofluidic chamber comprising the cell population comprising the oneor more cells that exhibit the extracellular effect, with amicrocapillary and aspirating the chamber's contents or a portionthereof to obtain a recovered aspirated cell population.

In one embodiment, if it is determined that a cell population comprisingone or more effector cells exhibit the extracellular effect, the cellpopulation or portion thereof is recovered to obtain a recovered cellpopulation, and the recovered cell population is further analyzed ascell subpopulations. The method, in one embodiment comprises retaining aplurality of cell subpopulations originating from the recovered cellpopulation in separate chambers of a microfluidic device, wherein eachof the separate chambers comprises a readout particle populationcomprising one or more readout particles, incubating the individual cellsubpopulations and the readout particle population within the chambers,and assaying the individual cell subpopulations for the presence of asecond extracellular effect. The readout particle population or asubpopulation thereof provide a readout of the second extracellulareffect and the second extracellular effect is the same extracellulareffect or a different extracellular effect as the extracellular effectmeasured on the recovered cell population. Once the cell subpopulationsare incubated and assayed, the method further comprises identifying,based on the results of the assaying step, a cell subpopulation fromamongst the plurality that comprises one or more cells that exhibit thesecond extracellular effect on the readout particle population, or asubpopulation thereof.

In another aspect, the present invention relates to a method ofidentifying a cell population displaying a variation in an extracellulareffect. In one embodiment, the method comprises, retaining a pluralityof individual cell populations in separate microfluidic chambers,wherein at least one of the individual cell populations comprises one ormore effector cells and the contents of the separate microfluidicchambers further comprise a readout particle population comprising oneor more readout particles, incubating the individual cell populationsand the readout particle population within the microfluidic chambers,assaying the individual cell populations for the presence of theextracellular effect, wherein the readout particle population orsubpopulation thereof provides a readout of the extracellular effect.Once the cell populations are incubated and assayed, the methodcomprises identifying, based on the results of the assay, a cellpopulation from amongst the plurality that exhibits a variation in theextracellular effect, as compared to one or more of the remaining cellpopulations of the plurality. In a further embodiment, the one or moreeffector cells comprise an antibody secreting cell. In anotherembodiment, the one or more effector cells comprise a plasma cell, Bcell, plasmablast, a cell generated through the expansion of memory Bcell, a hybridoma cell, a T cell, CD8+ T cell, and CD4+ T cell, arecombinant cell engineered to produce antibodies, a recombinant cellengineered to express a T cell receptor, or a combination thereof.

One or more cell populations exhibiting the extracellular effect orvariation in the extracellular effect, in one embodiment, are recoveredto obtain one or more recovered cell populations. Recovery, for example,is carried out with a microcapillary. Once one or more individual cellpopulations are identified and recovered, the one or more individualcell populations are further analyzed to determine the cell or cellsresponsible for the observed extracellular effect. In one embodiment,the method comprises retaining a plurality of cell subpopulationsoriginating from the one or more recovered cell populations in separatechambers of a microfluidic device. Each of the separate chamberscomprises a readout particle population comprising one or more readoutparticles. The individual cell subpopulations are incubated with thereadout particle population within the chambers. The individual cellsubpopulations are assayed for a variation of a second extracellulareffect, wherein the readout particle population or subpopulation thereofprovides a readout of the second extracellular effect. The secondextracellular effect is the same extracellular effect or a differentextracellular effect as the extracellular effect measured on therecovered cell population. Based on the second extracellular effectassay, one or more individual cell subpopulations are identified thatexhibit a variation in the second extracellular effect. The one or moreindividual cell subpopulations in one embodiment are then recovered forfurther analysis. Extracellular effect assays are described throughout.

In one embodiment, cells from a recovered cell population or recoveredcell subpopulation are retained in a plurality of vessels as cellsubpopulations or sub-subpopulations, and each cell subpopulation orcell sub-subpopulation is present in an individual vessel. Theindividual subpopulations or sub-subpopulations are lysed to provide andone or more nucleic acids within each lysed cell subpopulation or lysedcell sub-subpopulation are amplified. In a further embodiment, the oneor more nucleic acids comprise an antibody gene.

In one embodiment of the methods described herein, the incubating stepincludes exchanging the medium in the respective microreactors (e.g.,microfluidic chambers) comprising the individual cell populations orsubpopulations. Medium exchange is carried out, for example, to maintainthe viability of the cells in the chamber or to provide reagents forcarrying out an extracellular effect assay, or to perform multipleextracellular effect assays in a serial manner.

Incubating, in one embodiment, comprises incubating the cell populationsor cell subpopulations with a plurality of accessory particles. Theplurality of accessory particles is provided, for example, as additionalreagents for the extracellular effect assay or to maintain cellviability. In one embodiment, the plurality of accessory particlescomprises sphingosine-1-phosphate, lysophosphatidic acid, growth factor,cytokine, chemokine, neurotransmitter, virus particle, secondaryantibody, fluorescent particle, a fluorescent substrate, a complementpathway inducing factor, a virus particle or an accessory cell. Theaccessory cell, in one embodiment, is a fibroblast cell, natural killer(NK) cell, killer T cell, antigen presenting cell, dendritic cell,recombinant cell, or a combination thereof.

The extracellular effect measured by the methods and devices describedherein in one embodiment, is binding of an effector cell or moleculesecreted by an effector cell to a cell surface protein, antagonism of acell surface receptor, or agonism of a cell surface receptor present ona readout cell (a type of readout particle). In a further embodiment,the cell surface receptor is a receptor tyrosine kinase (RTK), aG-protein coupled receptor (GPCR), receptor serine-threonine kinase,receptor tyrosine phosphatase or a receptor guanylyl cyclase. The GPCRis not limited by class or species. For example, the GPCR, in oneembodiment, is a GPCR provided in Table 3A or 3B, herein.

In another embodiment, the extracellular effect measured by the methodsand devices described herein, is binding of an effector cell or moleculesecreted by an effector cell to an ion channel, antagonism of an ionchannel, or agonism of an ion channel. The ion channel, in oneembodiment, is a GABAA, Glycine (GlyR), serotonin (5-HT), nicotinicacetylcholine (nAChR), zinc-activated ion channel, ionotropic glutamate,AMPA, kainite, NMDA receptor or an ATP gated channel

Where the extracellular effect is binding, agonism or antagonism of acell surface receptor or ion channel, the effect in one embodiment ismeasured by detection of an increase in intracellular cAMP or calcium,expression of a protein reporter, or localization of a protein within areadout cell expressing the cell surface receptor or ion channel.

The extracellular effect in another embodiment, is a binding interactionbetween a molecule secreted by the one or more effector cells or asubset thereof, to one or more readout particles or one or moreaccessory particles, modulation of apoptosis, modulation of cellproliferation, a change in a morphological appearance of the readoutparticle, a change in localization of a protein within the readoutparticle, expression of a protein by the readout particle,neutralization of an accessory particle operable to affect the readoutparticle or a combination thereof.

In some embodiments, the extracellular effect is an effect of a cellproduct, secreted by an effector cell. The extracellular effect isbinding interaction between a protein produced by an effector cell andeither a readout particle or accessory particle. For example, theeffector cell in one embodiment is an antibody secreting cell (ASC), andthe readout or accessory particle comprises an epitope or an antigen.The binding interaction, in one embodiment, is a measure of one or moreof antigen-antibody binding specificity, antigen-antibody bindingaffinity, and antigen-antibody binding kinetics. In another embodiment,the effector cell is an activated T cell that secretes a cytokine, andthe readout particle includes one or more antibodies to capture thesecreted cytokines.

The above methods and devices may be used to screen or select for cellsare that may be rare, e.g. less than 1% of the cells in the population,or from about 1% to about 10% or from about 5% to about 10% of the cellsbeing screened or selected.

In another aspect, functional antibodies and receptors discoverable bythe methods herein are provided. In one embodiment of this aspect, thenucleic acid of an effector cell responsible for an extracellular effectis amplified and sequenced. The nucleic acid is a gene encoding for ansecreted biomolecule (e.g., antibody, or fragment thereof), or a geneencoding a cell receptor or fragment thereof, for example a T-cellreceptor. The antibody or fragment thereof or cell receptor or fragmentthereof can be cloned and/or sequenced by methods known in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a process flow diagram for one embodiment of a microfluidicapproach for single effector cell identification and selection based ona microfluidic multicellular assay. Single cells are obtained from anyanimal and are optionally enriched for an effector cell population.High-throughput microfluidic analysis is used to perform functionalscreens on antibodies secreted from single effector cells, in somecases, present in heterogeneous cell populations. After one or multiplerounds of microfluidic analysis, cells are recovered and antibodyvariable region genes are amplified for sequencing (Vh/Vl) and cloninginto cell lines. This process allows for the screening of over 100,000cells in a single day, with sequences recovered on week later.

FIG. 2 is a process flow diagram for one embodiment of a microfluidiceffector cell enrichment method. Effector cells are first loaded at anaverage concentration of 25 cells per chamber and incubated to createpolyclonal mixtures of antibodies. Screening of polyclonal mixtures isused to identify chambers having a variation in an extracellular effect(e.g., binding, affinity, or functional activity). Forty positivechambers are then recovered to achieve an enriched population with ˜4%of effector cells making antibodies of interest. The effector cells ofthe enriched population are then analyzed in a second array at limitingdilution to select a single ASC(s) having the variation in theextracellular effect. The time required for enrichment is about 4 hoursand total screening throughput is 100,000 cells per run. Enrichmentprocess may be performed twice if needed, and may use the same ordifferent properties for each screen.

FIG. 3 shows top and cross-sectional view schematic diagrams of a methodof identifying the presence of an effector cell that produces abiomolecule capable of specifically binding a target readout particleaccording to an embodiment of the invention.

FIG. 4 shows top and cross-sectional view schematic diagrams of a methodof identifying the presence of at least one effector cell that producesa biomolecule (e.g. antibody) that binds specifically to malignant cellsbut not normal cells.

FIG. 5 shows top and cross-sectional view schematic diagrams accordingto one embodiment of the invention of a method of identifying thepresence of an effector cell that produces a biomolecule that binds to areadout cell where a subpopulation of effector cells are functionalizedto also act as readout cells.

FIG. 6 is a schematic diagram of an antibody tetramer.

FIG. 7 shows top and cross-sectional view schematic diagrams of a methodof screening for a target epitope/molecule to which an known biomoleculebinds according to an embodiment of the invention.

FIG. 8 shows top and cross-sectional view schematic diagrams of a methodof identifying the presence of an effector cell which produces anantibody that specifically binds a target epitope/antigen according toan embodiment of the invention.

FIG. 9 shows top and cross-sectional view schematic diagrams of a methodof quantifying cell lysis.

FIG. 10 shows top and cross-sectional view schematic diagrams of amethod of identifying the presence of an effector cell which produces anantibody that specifically binds a target epitope/antigen according toan embodiment of the invention.

FIG. 11 shows top and cross-sectional view schematic diagrams of amethod of quantifying cell lysis.

FIG. 12 shows top and cross-sectional view schematic diagrams of amethod of identifying the presence of an effector cell which produces abiomolecule that induces growth of readout cells.

FIG. 13 shows top and cross-sectional view schematic diagrams of amethod of identifying the presence of an effector cell which produces abiomolecule that stimulates readout cells to undergo apoptosis.

FIG. 14 shows top and cross-sectional view schematic diagrams of amethod of identifying the presence of an effector cell which produces abiomolecule that stimulates autophagy in readout.

FIG. 15 shows top and cross-sectional view schematic diagrams of amethod of identifying the presence of an effector cell which produces abiomolecule that neutralizes a cytokine of interest.

FIG. 16 shows top and cross-sectional view schematic diagrams of amethod of identifying the presence of an effector cell which produces abiomolecule that inhibits the ability of a virus to infect a cell.

FIG. 17 shows top and cross-sectional view schematic diagrams of amethod of identifying the presence of an effector cell which produces abiomolecule that inhibits the function of a target enzyme according toan embodiment of the invention.

FIG. 18 shows top and cross-sectional view schematic of a method ofidentifying presence of an effector cell that displays a molecule thatelicits the activation of a second type of effector cell, which in turnsecretes molecules that have an effect on a readout particle.

FIG. 19 shows top and cross-sectional view schematic diagrams of amethod of identifying presence of an effector cell that secretes amolecule that elicits the activation of a second type of effector cell,which in turn secretes molecules that have an effect on a readoutparticle.

FIG. 20 shows top and cross-sectional view schematic diagrams of amethod to detect the presence of at least one effector cell secreting anantibody with high affinity from a heterogeneous population of cellscontaining cells that secrete an antibody for the same antigen but withlower affinity.

FIG. 21 shows top and cross-sectional view schematic diagrams of amethod of screening for antibodies with increased specificity for anantigen according to an embodiment of the invention in which readoutparticles displaying different epitopes are distinguishable by differentoptical characteristics.

FIG. 22 shows top and cross-sectional view schematic diagrams of amethod of simultaneously identifying the presence of a cell secreting abiomolecule in a homogeneous or heterogeneous population of effectorcells and analyzing one or more intracellular compounds affected by themolecule.

FIG. 23 is a top view schematic diagram of a method of evaluating theextracellular effect of an effector cell on multiple sets of readoutparticles simultaneously.

FIG. 24 is an alignment of the extracellular domain for PDGFRa acrosshuman, rabbit, mouse and rat. (Top) Ribbon diagram showing structure ofextracellular domain (ECD) of two PDGFRβ in complex with a dimer ofPDGFBB (from Shim et al. (2010). Proc. Natl. Acad. Sci. U.S.A. 107, pp.11307-11312, incorporated by reference herein in its entirety). Note, aPDGFRβ is shown since a similar structure for PDGFRα is expected but wasnot available. (Bottom) Alignment of ECD for PDGFRα across human (SEQ IDNO. 79), mouse (SEQ ID NO. 80), rabbit (SEQ ID NO. 81), rat (SEQ ID NO.82). Regions of variation from the human isoform are denoted by lightershading and “*”. The substantial variation indicates there are numerousepitopes available for antibody recognition, with rabbit having the mostvariation from human.

FIG. 25 provides images showing various aspects of multilayer softlithography microfluidics. (A) Optical micrograph of a valve made usingMSL. Two crossing microfabricated channels, one “flow channel” for theactive fluids (vertical) and one control channel for valve actuation(horizontal), create a valve structure. The flow channel is separatedfrom the control channels by a thin elastomeric membrane to create a“pinch valve”. Pressurization of the control channel deflects themembrane to close off the flow channel. (B) Section of a deviceintegrating multiple valves (filled with green and blue food dye). (C)Section of a device fabricated at UBC having a total of 16,000 valves,4000 chambers, and over 3000 layer-layer interconnects (arrow). (D)Example of a microfluidic device with penny for scale.

FIG. 26 is a schematic of one device of the invention. (A) Schematicshowing the structure of a microfluidic device for antibody selectionfrom single antibody-secreting cells. (B) Array of 4,032 analysischambers. Each chamber is isolated during incubation and media can beexchanged within minutes. (C) Close up of an individual chamber. Cells,readout particles and reagents are injected sequentially, settling downby gravity. Imaging is performed using automatedbrightfield/fluorescence microscopy.

FIG. 27 is a schematic of the layers that are assembled during oneembodiment of device fabrication.

FIG. 28 (A) Top view and side view of inflatable chamber design.Chambers have a circular geometry, with a larger circular “lip” at thetop, and are overlaid by a recess separated by a thin membrane. Valveconnecting chambers to a flow channel is sealed and cells are loadeddown the flow channel. (B) Valves to chambers are opened and pressure isapplied to inflate the chambers, causing cells to enter the tops ofchambers. (C) Valves are closed to seal chambers, and cells fall to thechamber floor. Channel is flushed with fresh medium. (D) Valves areopened and pressure is released, causing chambers to “deflate” back totheir original volume. Repetition of this process may be used toexchange medium and/or add soluble factors.

FIG. 29 is a schematic diagram of a microfluidic chamber having a cellfence according to an embodiment of the invention illustrating the useof laminar flow to direct particles to one side of the cell fence.

FIG. 30 is a schematic diagram of a microfluidic chamber having a cellfence according to an embodiment of the invention illustrating the useof a restriction upstream of the chamber inlet to preferentially directparticles to the one side of the inlet channel.

FIG. 31 is a schematic depiction of a reusable mold made from multiplelayers of photoresist on a silicon wafer substrate, which is used forPDMS microfluidic device fabrication.

FIG. 32 is a schematic diagram of a microfluidic chamber having a cellfence according to an embodiment of the invention illustrating the useof a cell fence that selectively separates particles based on particlesize.

FIG. 33 is a top view of a chamber embodiment with a series ofintersecting cell fences forming an array of wells, defining multipleeffector and readout zones, on the lower surface of the chamber (overallchamber dimensions are 300 μm×160 μm).

FIG. 34 is a drawing of particle trap embodiment (grid pitch is 2 μm)for a chamber having two perpendicular cell fences connected by agenerally circular portion defining a particle trap.

FIG. 35 is a schematic diagram of a microfluidic chamber according to anembodiment of the invention illustrating the use of micro-fabricatedstructures positioned at the outlet to retain particles of a certainsize in the chamber.

FIG. 36 is a side view schematic diagram of a microfluidic chambercomprising dead-end cups at the bottom of the chamber.

FIG. 37 is a side view schematic diagram of a chamber according to anembodiment of the invention in which structural elements are positionedin the flow channel to retain effector cells and readout particles.

FIG. 38 is a side view schematic diagram of a chamber according to anembodiment of the invention in which structural elements are positionedsequentially in a chamber to segregate and retain particles on the basisof size.

FIG. 39 is a top view schematic diagram of a serial, flow througharrangement of microfluidic chambers in which a porous membrane is usedto separate effector cells from readout particles.

FIG. 40 is a top view schematic diagram of a serial, flow througharrangement of microfluidic chambers in which a layer ofnon-functionalized beads is used to separate effector cells from readoutparticles.

FIG. 41 is a schematic diagram of a microfluidic chamber according to anembodiment of the invention illustrating the use of a magnetic field toposition particles within the chamber.

FIG. 42 is a schematic diagram of a method of separating particlesaccording to an embodiment of the invention using a magnetic field todirect one type of particle to which magnetic particles are coupled to aposition within a chamber while having little or no effect on particlesto which the magnetic particles are not coupled.

FIG. 43 is a cross-sectional view of a chamber embodiment with cellfence, where the chamber has been tipped to facilitate readout particle(bead) loading into the readout zone.

FIG. 44 is a schematic diagram of a microfluidic chamber having a cellfence according to an embodiment of the invention illustrating the useof rotation of the chamber to preferentially direct particles to the oneside of the fence.

FIG. 45 is a schematic diagram of a method of separating effector cellsand readout particles according to an embodiment of the invention usingdifferential density of the effector cell and readout particle.

FIG. 46 is a schematic diagram of a microfluidic chamber comprising anintegrated electrode according to an embodiment of the inventionillustrating the use of a dielectric field to position particles withinthe chamber.

FIG. 47 shows a top view schematic diagram of a chamber according to anembodiment of the invention in which effector cells and readoutparticles are introduced to the chamber via separate inlets.

FIG. 48 is a top view schematic diagram of a compound chamber accordingto an embodiment of the invention in which an effector zone subchamberand a readout zone sub chamber may be placed in fluid communication witheach other.

FIG. 49 is a schematic diagram of a method of separating effector cellsand readout particles using surface functionalization to confineanchorage-dependent readout cells in a readout zone at the chamberceiling and gravity to confine suspension effector cells on the bottomof the chamber.

FIG. 50 is a top view schematic diagram of a chamber functionalized tomaintain adherent cells on only one side while suspension cells aresegregated to the opposite side by gravity.

FIG. 51 is a top view schematic diagram of a chamber functionalized withtwo types of antibodies in order to segregate particles displayingdifferent types of antibodies on their surface.

FIG. 52 is a top view schematic diagram of a serial, “flow-through”arrangement of microfluidic chambers in which each chamber is isolatedfrom its neighbour by a valve positioned between the chambers.

FIG. 53 is a top view schematic diagram of a serial, “flow-through”arrangement of microfluidic chambers in which each chamber may beisolated from a flow channel shared by neighboring chambers by a “lid”structure.

FIGS. 54 and 55 are top view schematic diagrams of parallel,“flow-through” arrangements of microfluidic chambers in whichneighboring chambers may share a common inlet channel and outlet buschannel.

FIG. 56 is top and side view schematic diagrams of a “dead-end” filledchamber for used in a parallel arrangement of microfluidic chambers.

FIG. 57 is a schematic diagram of a parallel arrangement of dead-endfilled chambers which can provide compound chamber functionality.

FIG. 58 are images of a microfluidic instrument for cell recovery and animage sequence during cell recovery. Top: From left to right. Opticalmicrograph of image sequence during cell recovery with cells in chamber,capillary piercing chamber roof (far left), empty chamber followingaspiration, and capillary dispensing cells into tube (far right). Bottomleft: Image of custom-built microfluidic screening instrument including(i) Microcapillary mounted on robotic micromanipulator, (ii) digitalpneumatics for nanoliter flow aspiration/dispensing, (iii) X-Ytranslation mount, (iv) incubator insert with mounts for recovery tubes,(v) scanning X-Y stage for image acquisition across the array, (vi)inverted microscope, (vii) cooled Hamamatzu CCD camera forhigh-sensitivity fluorescent imaging, (viii) control solenoids forcapillary operation. Bottom right: Close up of microfluidic devicemounted beneath incubator insert with capillary positioned for cellrecovery. (C) Optical micrograph of image sequence during cell recoverywith cells in chamber (top left), capillary piercing chamber roof (topright), empty chamber following aspiration (bottom left), and capillarydispensing cells into tube (bottom right). (D) Performance of cellrecovery. When operating in Mode I, multiple chambers are aspiratedbefore dispensing. This is used for two-step screening (enrichment) ofcells or for recovery of pools of cells. Recovery from each chambertakes approximately 3 seconds. When operating in Mode II, the contentsof a single chamber are aspirated and dispensed followed by 4 washingsteps to ensure no carryover between single cells.

FIG. 59 is a schematic of single cell HV/LV approach usingtemplate-switching. Single cells are deposited into microfuge tubes andcDNA is generated from multiplexed gene-specific primers targeting theconstant region of heavy and light chains. Template-switching activityof MMLV enzyme is used to append the reverse complement of atemplate-switching oligo onto the 3′ end of the resulting cDNA.Semi-nested PCR, using multiplexed primers that anneal to the constantregion of heavy and light chain and a universal primer complementary tothe copied template switching oligo, is used to amplify cDNA andintroduce indexing sequences that are specific to each single cellamplicon. Amplicons are then pooled and sequenced.

FIG. 60 is a schematic of the traditional hybridoma approach.Splenocytes from immunized mice are fused with myeloma cells. At a lowefficiency, these fusions create viable “hybridomas” that can secretemAbs and can be grown in culture. Pools of hybridomas are grown andassayed to detect presence of antigen-specific cells, which are thensubcloned and expanded to generate sufficient mAbs for functionalscreening. This approach requires a suitable fusion partner and islargely restricted to use with mice or rats, although a proprietaryhybridoma technology has also been developed for rabbits. Typicalfusions result in less than 100 stable hybridomas and require ˜9 weeksfor culture and subcloning.

FIG. 61 is an image showing a cross-section view of the microfluidicchamber array contained within a thin membrane with labels indicatingcell culture chamber, valves, and channel connecting chambers.

FIG. 62 is a schematic of the chamber of the microfluidic device shownin FIG. 61.

FIG. 63 shows a photograph of a microfluidic device having 8,192chambers arranged in 4 sub-arrays of 2,048. The microfluidic chamberarray is located directly under the osmotic bath reservoir within a300-micron thick layer of elastomer.

FIG. 64 is a schematic of the microfluidic device shown in FIG. 62.

FIG. 65 is a schematic depiction of a microfluidic device that enablesthe segregation of effector and target cells.

FIG. 66 is a schematic depiction of a single unit cell of themicrofluidic device from FIG. 65.

FIG. 67 is a micrograph of a cross-section taken along the verticaldashed line of FIG. 66.

FIG. 68 is a micrograph of a cross-section taken along the horizontaldashed line of FIG. 66.

FIG. 69 is a series of schematic diagrams showing an embodiment for acytokine neutralization assay.

FIG. 70A is a top view light microscopy image of a chamber embodimentwith five effector cells shown in the right end of the effector zone(top) and four readout particles in the readout zone (bottom).

FIG. 70B is a top view fluorescence microscopy image of the chamberembodiment shown in FIG. 70A with some fluorescence associated with theeffector cells at the right end of the effector zone and fluorescenceassociated with the four readout particles in the readout zone.

FIG. 70C is a top view light microscopy image of a chamber embodimentwith one effector cell shown in the effector zone (left) and one readoutparticle in the readout zone (right).

FIG. 70D is a top view fluorescence microscopy image of the chamberembodiment shown in FIG. 70C with fluorescence associated with thereadout particle in the readout zone.

FIG. 70E is a top view light microscopy image of a chamber embodimentwith two effector cells in the effector zone (left) and six readoutparticles in the readout zone (right).

FIG. 70F is a top view fluorescence microscopy image of the chamberembodiment shown in FIG. 70E with some fluorescence associated with thereadout particles in the readout zone.

FIG. 71 is a schematic of the workflow for the capture and detection ofantibodies from antibody-secreting cells.

FIG. 72 Example of fluorescent and bright field images from the beadimmunocapture assay followed by time-lapse imaging of the clone for 4.5days.

FIG. 73 demonstrate robust cell culture of CHO antibody-secreting cellsin the microfluidic array compared to batch shake flasks and singlecells seeded in 96-multiwell plates.

FIG. 74A is a light micrograph of a microfluidic chamber into which aHyHEL5 hybridoma cell secreting an anti-lysozyme antibody has beenloaded (top panel) and a microfluidic chamber into which the HyHEL5hybridoma cell has not been loaded (bottom panel).

FIG. 74B is a light micrograph of the chambers shown in FIG. 74A intowhich 4B2 hybridoma cells secreting “background” antibodies that do notbind lysozyme have been loaded in addition to HyHEL5 cells.

FIG. 74C is a fluorescence micrograph of the chambers shown in FIG. 74Bafter incubation with fluorescent lysozyme.

FIG. 74D is a fluorescence micrograph of the chambers shown in FIG. 18Cafter incubation with fluorescent anti IgG antibodies.

FIG. 74E is a graph showing kinetics of antibody accumulation andrelease for the chambers depicted in FIG. 74C.

FIG. 74F is a graph of the fluorescence of 600 chambers containing a mixof HyHEL5 and 4B2 hybridoma cells incubated in the presence of Protein Aand 10 nM lysozyme in the growth media recorded over time.

FIG. 75A shows three representative examples of the lack of signal inchambers containing antibody secreting cells but no cell secretingantibodies against an antigen of interest.

FIG. 75B shows three representative examples of chambers in which asingle cell secreting an antibody against an antigen of interest isdetected among a background of multiple cells secreting antibodies thatare not specific to the antigen of interest.

FIG. 75C shows a histogram of the fluorescent signal in all chamberscontaining only hybridoma cells that do not secrete antibodies againstthe antigen of interest.

FIG. 75D shows a histogram of the fluorescent signal in chamberscontaining a mixture of HyHEL5 hybridoma cells producing antibodiesagainst an antigen of interest (hen-egg lysozyme) and DMS-1 hybridomacells producing an antibody against a different antigen.

FIG. 76A is a schematic of a single hybridoma cell (4B2) secreting anantibody against human CD45 on fixed K562 cells. Cells and beads arestained with a detection antibody following incubation.

FIG. 77B shows the mean fluorescence intensity of readout cells andbeads measured by automated image analysis for empty chambers andchambers containing a single hybridoma.

FIG. 77C, from left to right: Chamber with a single hybridoma cell.Bright field, fluorescent and merged images of anti-CD45 antibodystaining in the same chamber following an overnight incubation withtarget fixed K562 cells and a 2-hour incubation period with protein Abeads.

FIG. 77A is a schematic of an immunization and binding assays. Mice wereimmunized with live cells from an ovarian cancer cells (TOV21G).Antibody-secreting cells were sorted using FACS and were then injectedin the microfluidic device and incubated with readout cells (fixed andlive TOV21G cells) stained with CFSE. Antibody binding is visualizedusing a secondary labeled antibody.

FIG. 77B shows plasma and readout cells (live and fixed) after loadingon chip. Readout cells are stained with CFSE for identification.Antibody binding on the cell surface of live and fixed cells isvisualized with a secondary labeled antibody. Far right shows a negativechamber with very low signal on the readout cells.

FIG. 78 is an image showing cell survival and antibody-secretion byELISPOT of (A) mouse ASCs grown for 8 days, and (B) human ASCs grown for5 days. The number of cells plated per well is indicated.

FIG. 79 shows ASC selection from mice immunized with ovarian carcinomacells. (A) Plot showing fluorescence-activated cell sorting of mousespleen cells stained with PE anti-mouse CD138. Gating shows CD138+population. (B) ELISPOT showing antibody secretion from unsorted spleencontrol, CD138− control, and CD138+ population. The number of cellsplated per well is indicated. (C) Graph showing ELISPOT counts as %spots per cells plated for each population.

FIG. 80 shows ASC selection from rabbits immunized with influenza. (A)Plot showing fluorescence-activated cell sorting of rabbit PBMCs usingER-Tracker and mouse anti-rabbit IgG. Gating shows selection ofER^(high)IgG^(low) population. (B) ELISPOT showing antibody secretionfrom unsorted PBMCs control, ER^(high)IgG^(low) population, and no cellcontrol. The number of cells plated per well is indicated. (C) Graphshowing ELISPOT counts as % spots per cells plated for unsorted PBMCscontrol and ER^(high)IgG^(low) population.

FIG. 81A shows a representative example of bright field (top) andfluorescent (bottom) images from an antigen-specific positive chamberbefore enrichment.

FIG. 81B shows cells in a multiwell plate after culture and recoveryovernight.

FIG. 81C shows a representative example of bright field (top) andfluorescent (bottom) images of an antigen-specific positive chamberloaded at single-cell dilution after enrichment.

FIG. 81D the frequencies of H1N1- and H3N2-positive chambers before andafter enrichment.

FIG. 82 is a light microscopy image showing 2 chambers, one containingmultiple effector cells with at least one of them secreting an antibody(top) and another chamber without any effector cell (bottom). Thereadout particles form aggregates when secreted antibodies present (top)and remain dispersed in the absence of antibody-secreting effector cell(bottom).

FIG. 83 is a fluorescence microscopy image showing the two chambers in16G, one containing multiple effector cells with at least one of themsecreting an antibody (top) and another chamber without any effectorcell (bottom). Both chambers contain readout particles (protein A beads)that have been stained with a fluorescently labelled anti-human antibodyto determine the presence of an extracellular effect.

FIG. 84A is a diagram of the experiment depicted in Example 13.

FIG. 84B are optical micrographs of microfluidic chambers havingdifferent concentrations of labeled antigen.

FIG. 84C is a graph of bead fluorescent intensities at differentconcentrations of labeled antigen (hen-egg lysozyme) after incubationwith single hybridoma cells (HyHEL5 and D1.3) secreting antibodies withdifferent affinities.

FIG. 84D is a graph showing the bead fluorescent intensitiescorresponding to images in FIG. 84B after incubation with single D1.3and HyHEL5 cells secreting antibodies with different affinities andafter labeling with different concentrations of antigen (hen egglysozyme).

FIG. 85 show a section of a microfluidic array containing human plasmacells secreting antibodies against H3N2 after incubation with a closedvalve that maintained each chamber isolated.

FIG. 86 show a section of a microfluidic array containing human plasmacells secreting antibodies against H3N2 after incubation without usingthe isolation valve, allowing chambers to remain connected by the flowchannels.

FIG. 87 shows representative examples of affinity measurements obtainedby microfluidic screening for two single primary mouse plasma cellsproducing antibodies against hen-egg lysozyme.

FIG. 88 is a bar graph indicating that the remaining fluorescence levelof beads in HyHEL5-positive chambers is higher than in the rest of thechambers at the end of the wash.

FIG. 89A is a top view light microscopy example of a chamber containinga heterogeneous population of cells (erythrocytes and human B cells).

FIG. 89B is a top view light microscopy example of a chamber containinga heterogeneous population of cells and a population of readoutparticles (protein A beads).

FIG. 89C is a top view fluorescence microscopy example of a chambershowing that at least one cell in the heterogeneous population secreteshuman IgG antibodies. The antibody was captured by the readoutparticles, which were stained with Dylight594-conjugated anti-humanantibodies.

FIG. 89D is a top view light microscopy example of a chamber with aheterogeneous population of cells and a population of readout particlesafter flowing the H1N1 antigen into the chamber.

FIG. 89E is a top view fluorescence microscopy example of aheterogeneous population of cells in conjunction with a population ofreadout particles (protein A beads) after flowing the H1N1 antigen intothe chamber. The H1N1 antigen was conjugated to Dylight 488 so as todifferentiate antigen-specific staining from whole IgG staining.

FIG. 89F is a top view light microscopy example of a chamber afterrecovery of the cells.

FIG. 90 shows an example of a chemiluminescent signaling assay usingPathHunter® eXpress CCR4 CHO-K1 β-Arrestin GPCR Assay in multiwellplates.

FIG. 91 is a schematic representation of the experiment. Humanvolunteers were immunized with the seasonal flu vaccine Peripheral bloodmononuclear cells (PBMCs) were recovered and sorted with flow cytometryto enrich for plasma cells. The cells were injected in the microfluidicdevice and assayed for H1N1 and H3N2 specificity.

FIG. 92 is an example of a single human plasma cell in a chamber withprotein A beads. The secreted antibody captured on the beads binds toboth H1N1 and H3N2 labeled antigens and is therefore cross-reactive.Labeled anti-human IgG allows visualization of total IgG secretion.

FIG. 93 shows an example of a primary human antibody-secreting cell (topleft) identified as producing an antibody against influenza using a beadassay (bottom left) and having divided during an overnight incubation inthe microfluidic device (top right).

FIG. 94 is a picture of a Size Select® 2% agarose gel of antibody heavyand light chain gene specific PCR products after single cell screeningin a microfluidic device. Lanes i to iv show the products of heavy chainPCR amplification of samples 3 to 6, respectively. Lane v shows thenucleic acid ladder. Lanes vi to viii show kappa chain PCR amplificationof samples 3, 5, and 6, respectively. Lane ix shows lambda chain PCRamplification of sample 4.

FIG. 95A is a gel showing the amplification of both heavy and lightchains from two single cells secreting antibodies against influenza.

FIG. 95B shows the variable heavy and light amino acid sequences from 2cells secreting antibodies (Hs7 antibody and Hs15 antibody) against H1N1and H3N2. Hs7 heavy chain amino acid sequence: SEQ ID NO: 10, Hs7 lightchain amino acid sequence: SEQ ID NO: 12; Hs15 heavy chain amino acidsequence: SEQ ID NO: 14, Hs15 light chain amino acid sequence: SEQ IDNO: 16.

FIG. 95C is the functional validation of recombinant human mAbs thatcross-react with both H1N1 and H3N2.

FIG. 96A shows an example of a chamber with a heterogeneous populationof rabbit plasma cells containing at least one effector cell secretingan antibody against H1N1 detected by a fluorescent signal on readoutcapture beads.

FIG. 96B shows an example of a chamber with a heterogeneous populationof rabbit plasma cells containing at least one effector cell secretingan antibody against H3N2 detected by a fluorescent signal on readoutcapture beads.

FIG. 97A shows bright field images of 4 chambers loaded with a pluralityof enriched rabbit plasma cells

FIG. 97B shows fluorescent images of the chambers in FIG. 123A afterH1N1 detection. All chambers are negative and do not contain cellssecreting antibodies against H1N1.

FIG. 97C shows fluorescent images of the chambers in FIG. 123C afterH3N2 detection. Chambers exhibit variable bead intensities but all ofthem are positive and contain at least one cell secreting antibodiesagainst H3N2.

FIG. 97D is a gel showing the heavy and light chains amplified fromrabbit cells after recovery from the H3N2-positive microfluidic chambersin FIG. 123C.

FIG. 98 shows an image of the capillary loaded with recovered cellsapproaching the injection port immediately before re-injection forenrichment

FIG. 99 shows an example of the validation of human antibody sequencesby cloning, expression and characterization of the antibodies

FIG. 100 is a gel showing bands from RT-PCR amplification of hybridomasingle cells recovered from a microfluidic device.

FIG. 101 is a graph that compares the affinities of anti-hen egglysozyme antibodies produced by hybridomas (D1.3 and HyHEL5) andrecombinant expression of the sequences retrieved from single D1.3 andHyHEL5 hybridoma cells screened in a microfluidic device. Anti-mouseantibody capture beads were incubated with cell supernatants fromhybridomas or recombinant HEK293 cells expressing D1.3 and HyHEL5antibodies, washed and incubated with different concentrations of thelabeled antigen. Fluorescent measurements were normalized to the maximumbead intensity at the highest antigen concentration to validate thebinding properties of the recombinantly produced antibodies.

FIG. 102 is a graph that shows the fluorescent intensity of beadsincubated with the supernatant from HEK293 cells (control) or HEK293cells transiently expressing the antibody R05C14, followed by labeledhen-egg lysozyme (10 nM). The binding of a novel mouse antibody tohen-egg lysozyme was confirmed after the sequence R05C14 was obtainedfrom a primary mouse plasma cell identified as antigen-specific in amicrofluidic screen.

FIG. 103A is an image of a PCR gel showing the amplicons produced by themethods described in Example 25 using a gradient of RT temperaturesranging from 60° C. to 40° C.

FIG. 103B shows the results of Sanger sequencing of the band from 400 to600 bp shown in FIG. 103A. The sequence was aligned and confirmed tomatch the variable region sequence of the heavy chain of D1.3.

FIG. 104A-C are schematic representations of a method for the functionalinterpretation of the IgG repertoire based on next-generationsequencing.

FIG. 105A is a schematic of antigen detection multiplexing using beadsof different fluorescent intensities

FIG. 105B is a bright field image of three types of readout beads loadedin microfluidic chambers.

FIG. 105C is a fluorescent image of three types of readout beads withdifferent intensities and antigens in microfluidic chambers.

FIG. 105D is a fluorescent image of three types of readout beads afterdetection with a rabbit anti-H1N1 antibody and a secondary anti-rabbitantibody, with only H1N1-coated beads (arrows) displaying a signal.

FIG. 105E shows the signal after H1N1 detection on three types of beadscoated with different influenza strains and distinguished based on theirfluorescent intensities.

FIG. 106A shows the toxicity response of L929 cells in the presence ofactinomycin-D as a function of TNF-α concentration.

FIG. 106B shows an apoptosis and necrosis assay using L929 cellscultured in a microfluidic device in the presence of TNF-α andactinomycin-D.

FIG. 107A shows time-lapse fluorescence microscopy images from TNFαfunctional assay. (A) Upper panel: In the absence of TNFα ligandfluorescence localization is cytoplasmic. Middle panel: Upon activationby TNFα ligand 10 ng/mL, a change in fluorescence from cytoplasmic tonuclear is observed. Lower panel: In the presence of cell supernatantcontaining an antibody that neutralizes TNFα ligand in addition to TNFαligand 10 ng/mL, the fluorescence localization remains cytoplasmic.

FIG. 107B is a plot showing frequency of activated cells exhibitingnuclear fluorescence localization. The number of cells quantified isindicated, n.

FIG. 108 shows optical micrographs at 0 days, 1 day and 3 days of SKBR3cell populations in an individual microfluidic chambers. SKBR3 cellsincluded an LC3-GFP reporter.

FIG. 109A shows a bright field image of a chamber containing apopulation of peripheral blood mononuclear cells incubated in thepresence of IFNγ capture beads after activation with CEF peptides.

FIG. 109B shows a fluorescent image of a chamber containing at least oneactivated T cell secreting IFNγ after activation with CEF peptides.

FIG. 109C shows a bright field image of a chamber containing a T cellclone cultured for 5 days after activation with CEF peptides.

FIG. 109D shows a higher sensitivity using the microfluidic assaycompared to ELISPOT to measure number of antigen-specific T cells in apopulation of peripheral blood mononuclear cells stimulated with CEFpeptides.

FIG. 110 are fluorescence microscopy images of chambers from 3 subarraysfrom a cell survival PDGFRα functional extracellular effect assay,showing YFP fluorescence readout in BaF3 clone expressing PDGFRα andhistone 2B-YFP in the presence of (A) no ligand or (B) PDGF-AA 25 ng/mLfor T=48 hours. Insets show close-up of individual microfluidicchambers. FIG. 110C shows micrographs of a population of enriched mousesplenocytes (2 cells, black arrow) co-cultured with a population of livereadout cells (BaF3 overexpressing PDGFRA, white arrows) and containingat least one effector antibody-secreting cell after 12, 24, 36 and 48hours of culture in a microfluidic device.

FIG. 111 shows fluorescent images obtained from a plate-based assay inwhich PathHunter® eXpress CCR4 CHO-K1 β-Arrestin GPCR reporter cellswere incubated with different concentrations of the agonist CCL22,followed by different concentrations of the substrate C₁₂FDG. Activationof the GPCR CCR4 caused complementation of the β-galactosidase enzyme,which in turn cleaved the substrate into a fluorescent product.

FIG. 112A shows bright field and fluorescent images of amicrofluidic-based GPCR signaling assay in which in which PathHunter®eXpress CCR4 CHO-K1 β-Arrestin GPCR reporter cells were loaded in amicrofluidic device, incubated with the C₁₂FDG substrate for 90 minute,followed by incubation with different concentrations of the agonistCCL12 for 90 min. Activation of the GPCR CCR4 by the agonist causedcomplementation of the β-galactosidase enzyme, which in turn cleaved thesubstrate into a fluorescent product.

FIG. 112B is a graph representing the fluorescent intensity measurementsof PathHunter® eXpress CCR4 CHO-K1 β-Arrestin GPCR reporter cellsincubated with the substrate C₁₂GDF and different concentrations of theagonist CCL12, as shown in FIG. 112A.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a thing” includes more than one such thing.Citation of references herein is not an admission that such referencesare prior art to the present invention.

“Readout,” as used herein, refers to the method by which anextracellular effect is reported. A “readout particle population” cancomprise one or more readout particles, as described herein.

“Extracellular effect,” as used herein, is a direct or indirect effecton a readout particle that is extracellular of an effector cell,including but not limited to increased cellular proliferation, decreasedgrowth, apoptosis, lysis, differentiation, infection, binding (e.g.,binding to a cell surface receptor or an epitope), morphology change,induction or inhibition of a signaling cascade, enzyme inhibition, viralinhibition, cytokine inhibition, activation of complement. As providedherein, the extracellular effect in one embodiment is the binding of abiomolecule of interest, secreted by an effector cell, to a readoutparticle. In another embodiment, the extracellular effect is a responsesuch as apoptosis of a readout cell or accessory cell.

The methods provided herein are used to identify an effector cell orcell population comprising an effector cell(s) that displays a variationin an extracellular effect. The variation in the extracellular effect isa variation compared to a control (negative or positive control), or avariation compared to one or more of other cell populations.

A “heterogeneous population” as referred to herein, particularly withrespect to a heterogeneous population of particles or cells, means apopulation of particles or cells that includes at least two particles orcells that have a differing feature. For example, the feature in oneembodiment is morphology, size, type of fluorescent reporter, adifferent cell species, phenotype, genotype, cell differentiation type,the sequence of one or more expressed RNA species or a functionalproperty.

A “subpopulation,” as referred to herein, means a fraction of a greaterpopulation of particles (cells). A population of cells in one embodimentis divided into subpopulations, for example, by isolating individualsubpopulations in individual microfluidic chambers. Additionally, anindividual subpopulation can be partitioned into further subpopulations,for example, in a plurality of microfluidic chambers or other reactionvessels. A subpopulation may also be a fraction of particles within agreater population, located in the same microfluidic chamber. Asubpopulation contains one or more particles, and where a plurality ofparticles are present in a subpopulation, the individual particleswithin the plurality can be homogeneous or heterogeneous with respect toone another.

A “cell retainer” in one embodiment, defines at least one effector zoneand at least one readout zone either continuously or intermittently. Theretainer may be a structural element such as a valve, a cell fence, theorientation in an external field or field gradient (e.g., gravitational,magnetic, electromagnetic, acceleration, etc.), the orientation and/orlocalization of a locally generated field by an electrode or opticalcomponent or magnetic probe, surface modifications (for exampletexturing, coatings, etc.) that facilitate or inhibit cell adhesion, orby the specific gravity of a solution within the chamber, or may beachieved by a combination of one or more of the preceding.

“Coating” as used herein may be any addition to the chamber surface,which either facilitates or inhibits the ability of an effector cell ora readout particle to adhere to a surface of the chamber. The coatingmay be selected from one or more of the following: a cell; a polymerbrush; a polymer hydrogel; self assembled monolayers (SAM),photo-grafted molecules, a protein or protein fragment having cellbinding properties (for example, a cell binding domain from actin,fibronectin, integrin, protein A, protein G, etc.). More generallyArginine-glycine-aspartate-(serine) (RGD(S)) peptide sequence motif areused. Poly-L-Lysine is also widely used as a polymer coating with PDMSto enhance cell adhesion via electrostatic interactions; a phospholipidhaving cell binding properties, a cholesterol having cell bindingproperties, a glycoprotein having cell binding properties and aglycolipid having cell binding properties. In addition PDMS surfacefunctionalization using biotinylated biomolecules is a simple, highlyattractive and yet flexible approach. It is widely known that bovineserum albumin (BSA) due to hydrophobic domains readily adsorbs viahydrophobic effect on hydrophobic PDMS surfaces enabling further directcoupling of streptavidin based conjugates in the chambers (protein, DNA,polymers, fluorophores). Polyethylene glycol based polymers are alsoknown for their bio-fouling properties and can be coated on PDMS surface(adsorption, covalent grafting), preventing cell adhesion.Poly(paraxyxlylene), e.g., parylene C can also be deposited usingchemical vapor deposition (CDV) on PDMS surfaces and prevent cellularadhesion.

“Isolated,” as used herein, refers the circumstances under which a givenchamber does not permit substantial contamination of an effector celland/or readout particle being analyzed with a particle(s) orbiomolecule(s) of another chamber of the microfluidic device. Suchisolation may be achieved, for example, by sealing a chamber or a set ofchambers in the case of compound chambers, by limiting fluidcommunication between chambers or by restricting fluid flow betweenchambers.

An “inlet” or an “outlet,” as used herein, includes any aperture wherebyfluid flows into and out of a chamber. Fluid flow may be restrictedthrough the inlet or outlet or both to isolate a chamber from itssurrounding environment. There may be one or more valves to controlflow, or flow may be controlled by restricting the fluid channels, whichlead to the inlets and outlets with a layer which prevents flow (forexample, a control layer or isolation layer). Alternatively, flow may beregulated by the rate at which fluids are passed through the device. Theinlet or outlet may also provide fluid flow to the device for thedelivery of an effector cell or readout particle, or other componentscarried in the flow as needed during analysis. In some embodiments, theinlet and outlet may be provided by a single aperture at the top of thechamber over which fluid flows from an inlet side to an outlet side.

A “magnet,” as used herein, includes any ferromagnetic or paramagneticmaterial. “Ferromagnetic” as used herein is meant to include materialswhich may be comprised of iron, nickel, chromium, or cobalt orcombinations thereof and various alloys, such that the magnetic materialis attracted to a magnet or is a magnet itself. For example, a magneticmaterial may be made from ferromagnetic stainless steel or may be madefrom a rare earth magnet or a stainless steel having magneticproperties. A magnet may also be made by use of a ferrofluid in adefined shape or orientation. A magnet may also be implemented using acoil or other electrically actuated device designed to generate amagnetic field when energized with electrical current.

An “array of wells,” as used herein, refers to any array of structureswithin a chamber that may limit the movement of an effector cell and/ora readout particle by localizing one or the other to a particularreadout zone or effector zone, whereby a zone (i.e., either effector orreadout) may be defined by what type of particle resides within it. Forexample, one embodiment of an array of wells is shown in FIG. 33, wherea series of intersecting cell fences form an array of wells on a surfaceof the chamber.

A “particle trap,” as used herein, refers to a structure that is capableof spatially confining an effector cell or a readout particle(s)(bead(s)) to a specific spatial position to limit movement during anassay. A “cell fence” is one type of “particle trap.” In one embodiment,a particle trap is used to confine effector cells, in which case it maybe referred to as an “effector cell trap.” In some embodiments, aparticle trap will be used to confine readout particles, in which caseit may be referred to as a “readout particle trap”, or a “readout celltrap”, as the case may be. A “particle trap,” in one embodiment, allowsfor the particle being trapped to have a fixed position. Having a fixedposition may simplify imaging and image analysis. Having a fixedposition for a readout particle or particles may also have the advantageof limiting or controlling the diffusion distance between an effectorcell and a readout particle. Having a fixed position may also preventinteraction of effector cells and readout particles.

A “textured surface,” as used herein, may be any type of surfacemodification that would promote or reduce cell adhesion to the chambersurface. For example, the surface may be textured with one or more of:bumps, indentations, roughness, protrusions, hooks, pegs, wells,grooves, ridges, grain, weave, web, hydrophobicity, hydrophillicity,etc.

The human body makes millions to billions of different types ofantibodies at any given time, each produced by a different single plasmacell called an “antibody secreting cell” or “ASC.” Each ASC has adiameter of from about 7 μm about 15 μm, depending on source, a diameterapproximately 1/10^(th) the width of a human hair, and generates only aminute amount of antibody. Of the billions of different ASCs in thehuman body, only a very rare few make an antibody that is suitable to beused as a therapeutic. When analyzed in the volume of conventionalformats this small amount of antibody is too dilute, making itcompletely undetectable. For this reason antibody discovery currentlyrequires that each ASC be isolated, fused to an immortal cancer cell tocreate a hybridoma and “grown,” ultimately generating many thousands ofidentical cells that can produce enough antibody to be measured (seeFIG. 60). For example, see McCullough and Spier (1990). MonoclonalAntibodies in Biotechnology: Theoretical and Practical Aspects, Chapter2, Cambridge University Press, incorporated by reference herein in itsentirety). This process is not only incredibly inefficient (99.9% of thestarting immune cells are lost) but also very slow and expensive,requiring at minimum 3 months of labor before therapeutic function canbe tested. As a result, the discovery of antibodies with optimaltherapeutic properties is a major and unresolved bottleneck in drugdevelopment.

ASCs are terminally differentiated cells that cannot be directlyexpanded in culture. Existing methods for overcoming this issue as setforth above (e.g., the hybridoma method, see FIG. 60) are veryinefficient, capturing only a tiny fraction of the antibody diversity(typically <0.1%). These approaches are also restricted to use withrodents and are very slow and expensive, requiring months of laborbefore therapeutic function can be tested. As explained below, thepresent invention overcomes these limitations by allowing for directfunctional assays on antibodies and ASCs, regardless of source.

A variety of technologies have been advanced to increase the speed andthroughput of antibody screening, but these technologies do so at theexpense of information. Specifically, existing technologies arerestricted to the selection of antibodies based on binding, affinity andspecificity. While sufficient for research applications, manytherapeutic applications require high affinity antibodies that do morethan just bind to the target. Rather, therapeutic applications requireantibodies that induce the desired biological response (e.g.,agonists/antagonists of cell signaling; activation of immune responses;induction of apoptosis; inhibition of cellular growth ordifferentiation). Presently, all high-throughput antibody discoverytechnologies require that this functional characterization be performeddownstream, after binding of a target is assessed, using methods thatare cumbersome, costly, and low-throughput, even as compared to thehybridoma approach. For this reason the hybridoma method, developed over40 years ago, is still a mainstay in therapeutic antibody discovery.

The present invention in one aspect, harnesses the small reactionvolumes and massively parallel assay capabilities of a microfluidicplatform in order to screen cell populations for a property of interest,referred to herein as an “extracellular effect.” Each cell populationoptionally comprises one or more effector cells. The extracellulareffect is not limited to a particular effect; rather, it may be abinding property (specificity, affinity) or a functional property, forexample agonism or antagonism of a cell surface receptor. In oneembodiment, the extracellular effect is an effect exerted by a secretionproduct of a particular effector cell.

The integrated microfluidic devices and methods provided herein arebased in part on the concept that small is sensitive—each devicecomprises many thousands of nanoliter volume cell analysis chambers,each approximately 100,000 times smaller than conventional plate-basedassays. In these small nanoliter chambers, each single effector cellproduces high concentrations of secreted biomolecule within minutes. Forexample, each sing ASC produces high concentrations of antibodies withinminutes. This concentration effect, in one embodiment, is harnessed toimplement cell-based screening assays that identify antibodies, made bysingle primary ASCs, with a specific functional property(ies), such asthe modulation (e.g., agonism or antagonism) of cell surface receptoractivity. Functional assays amenable for use with the methods anddevices provided herein are described in detail below. Importantly, thein the screening methods provided herein, it is not necessary that aspecific effector cell or subpopulation of effector cells, having theparticular property be identified so long as the presence of theextracellular effect is detected within a particular microfluidicchamber comprising a cell population. Some or all of the cells withinthe chamber where the effect is measured can be recovered for furthercharacterization to identify the specific cell or cells responsible forthe extracellular effect. By completely eliminating the need for cellculture prior to screening, the single-cell approach provided hereinenables, for the first time, the direct selection of functionalantibodies from any species, in only days, and at a throughput ofgreater than 100,000 cells per run (FIG. 1).

The microfluidic devices and methods provided herein provide advantagesover currently available strategies for assessing an extracellulareffect of a single cell, for example, an extracellular effect of anantibody secreted by a single ASC. For example, the devices describedherein are scalable, enable reduced reagent consumption and increasedthroughput to provide a large single cell assay platform for studiesthat would otherwise be impractical or prohibitively expensive.Moreover, currently available single cell assay platforms analysesrequire multiple cell handling and processing steps in conventionaltubes in order to generate products needed for downstream analysis, forexample qPCR. The inclusion of microfluidic cell handling and processingas described herein thus offers important avenues to improved throughputand cost, while also improving precision and sensitivity throughsmall-volume confinement.

Without wishing to be bound by theory, the concentration enhancement andrapid diffusive mixing afforded by the nanoliter microfluidic chambersprovided herein, along with precise cellular handling and manipulation(e.g., spatio-temporal control of medium conditions) enables the singlecell analysis of effector cells such as immune cells (e.g., B cells, Tcells, and macrophages) whose primary functions include the secretion ofdifferent effector proteins such as antibodies and cytokines.

Embodiments described herein provide microfluidic systems and methodscapable of performing multicellular assays of secreted products fromcell populations comprising one or more effector cells, followed byrecovery of the cell populations for subsequent analysis. In someembodiments, the cell populations are heterogeneous cell populations.That is, two or more cells in a population differ in genotype, phenotypeor some other property. Moreover, where cell populations are assayed inparallel on one device, at least two of the populations areheterogeneous with respect to one another (e.g., different number ofcells, cell type, etc.). In the assays described herein, a readoutparticle population comprising one or more readout particles, whichserve as detection reagents (e.g., readout cells expressing a cellreceptor, readout bead, sensor, soluble enzyme, etc.) are exposed to acell population comprising one or more effector cells, and secretedproducts from the one or more effector cells, at a sufficientconcentration for a readout signal (e.g., a fluorescent signal) to bedetected. In some embodiments, the readout signal reports a biologicalresponse/functional effect (e.g., apoptosis) induced by one or more theeffector cells in the population on one or more readout particles (e.g.,readout cells). For example, for an antibody produced from a given ASC,the ASC or a cell population comprising one or more ASCs, along withreadout particle(s) and optionally accessory detection reagents aresequestered in a small volume so on a device, and the assay is carriedout (chambers having a volume of about 100 μL to about 50 nL, e.g. about1 nL to about 5 nL). Importantly, because effector cells in oneembodiment are rare cells, not all cell populations assayed by themethods described herein will initially contain an effector cell. Forexample, where thousands of cell populations are assayed on a singledevice, in one embodiment, only a fraction of the chambers will comprisean effector cell. The methods provided herein allow for theidentification of the chambers with the effector cell.

The present invention takes a different approach than previouslydescribed microfluidic methods. The latter take the approach of loadingsingle cells at a density to maximize the number of single cells inindividual chambers (for example, where droplets or microwells areused). This is accomplished by isolating single cells by limitingdilution followed by analysis of the fraction of chambers or volumesthat contain a single cell. Such a strategy sacrifices throughputbecause the optimal single cell loading is achieved at approximately 1cell per well average density. Similarly, the geometries described forthese methods usually do not allow for more than a few cells in achamber and in many cases are designed to physically accommodate only asingle cell. Besides a decreased throughput, a number of technicalchallenges result from the approach of isolating and assaying a singlecell in a single microfluidic chamber. For example, achieving sufficientcell concentrations to achieve a meaningful readout from heterogeneouspopulations, keeping individual cells alive due to nutrient depletion,the need for aeration, unwanted vapor permeation effects, poorlycontrolled medium conditions, and the need for waste removal, can poseserious problems for achieving a reliable reproducible single cellmicrofluidic assay. In many functional assays, like cell growthinhibition, it is necessary to keep both the effector cells and thereadout cells alive for several days. Such an assay is not feasible inmicrowell based systems and droplets. However, as discussed herein, thepresent invention provides a robust platform for assays spanning days.

The devices and assays described herein provide single cell assayswhereby one or more effector cells are present in individual cellpopulations, in single microfluidic chambers. The cell populations areassayed for their respective ability to exert an extracellular effect ineach chamber, thereby providing a higher total throughput thanpreviously described methods. Importantly, effects of a single effectorcell can be detected within a larger cell population, for example aheterogeneous cell population. By taking the multicellular assayapproach within a single microfluidic chamber, the embodiments describedherein can operate at greater than or equal to 100 times the throughputreported previously. Once a cell population is identified that anextracellular effect on a readout particle, or a variation in anextracellular effect as compared to another population, the cellpopulation, in one embodiment, is recovered and further assayed asindividual cell subpopulations (e.g., the recovered cell population isassayed at limiting dilution) to determine which effector cell(s) withinthe population is responsible for the extracellular effect.

Methods and apparatuses known in the art which are designed toaccommodate more than a single cell have limitations that make themunsuitable for the types of assays described herein, for example,maintaining cells in a viable state, the inability to selectivelyrecover effector cells of interest, evaporation within a device,pressure variability, cross-contamination, device architecture thatlimits imaging capabilities (e.g., by providing particles in differentfocal planes, reduced resolution) and lack of throughput (WO 2012/072822and Bocchi et al. (2012), each incorporated by reference in theirentireties).

Embodiments described herein relate in part to functional effector cellassays (also referred to herein as extracellular effect assays) thatallow for the detection of a single effector cell of interest present inan individual microfluidic chamber in a heterogeneous cell population.Specifically, in the case where a chamber contains a heterogeneous cellpopulation, where each cell of the population secretes antibodies (i.e.,a heterogeneous ASC population within a single microfluidic chamber), oronly a fraction of the cells in the population secrete antibodies,whereby only one effector cell or a subpopulation of effector cellssecretes an antibody that produces a desired extracellular effect on areadout particle(s), the embodiments described herein provide a methodfor measuring and detecting the desired extracellular effect. Once achamber is identified that comprises a cell population exhibiting theeffect, the population is recovered for downstream analysis, forexample, by splitting the cell population into subpopulations atlimiting dilution. As described below, in one embodiment, one or moreheterogeneous populations of cells displaying an extracellular effect,are recovered and subjected to further screening at limiting dilution(e.g., from 1 to about 25 cells per assay), to determine which cell theextracellular effect is attributable to.

In one embodiment, a microfluidic assay is carried out on a plurality ofcell populations, present in individual microfluidic chambers, todetermine whether an effector cell within one of the populationssecretes an antibody or other biomolecule that inhibits the growth of areadout cell. In this embodiment, even in the presence a heterogeneouscell population comprising a plurality of ASCs that secrete antibodieswhich do not affect the growth of the readout cell, the readout cellgrowth is still equally inhibited and the microfluidic chamber isidentifiable as containing the desired effector cell and secretionproduct. The chamber's contents can then be recovered for furthermicrofluidic analysis, or benchtop analysis, for example, at limitingdilution of the effector cells to determine which effector cell displaysthe effect. Antibody sequences can also be recovered by methods known tothose of skill in the art.

In one embodiment, novel antibodies are provided by the methodsdescribed herein. For example, one or more ASCs can be identified by themethods described herein, recovered, and their antibody genes sequencedand cloned.

Where single cells are loaded into individual chambers at a density ofapproximately 1 cell per chamber, the devices provided herein allow forthe screening of approximately 1000 single cells (e.g., ASCs) perexperiment. One or more of the single cells can be ASCs or a differenttype of effector cell. Although approximately 10-fold higher thanhybridoma methods, it is desirable in many instances to screen tens ofthousands, or even hundreds of thousands of cells. Examples of thisinclude when ASCs cannot be obtained at high purity (e.g., for speciesfor which ASC markers/antibodies are not available or cases of poorimmune response), or when antibodies that bind are frequent but thosewith desired properties are exceedingly rare (such as blocking of areceptor). However, after identifying a cell population(s) that containone or more effector cells displaying the extracellular effect ofinterest, the cell population(s), in one embodiment, are analyzed again,but at limiting dilution, e.g., as single cells in individualmicrofluidic chambers, or smaller populations in individual chambers (ascompared to the first screen), in order to determine the identity of theindividual effector cell(s) responsible for the extracellular effect.One embodiment of this two step screening method is shown in FIG. 2.Once the effector cell(s) is identified, its genetic information can beamplified and sequenced. In one embodiment, the genetic informationcomprises a novel antibody gene.

In the embodiment shown in FIG. 2, a microfluidic array is loaded at adensity of approximately 25 cells per chamber, resulting in a total ofapproximately 100,000 cells in a single device. The chambers are thenisolated and incubated, generating unique polyclonal mixtures ofantibodies in each chamber. These antibodies are then screened toidentify chambers that exhibit the desired extracellular effect, e.g.,antigen binding, high binding affinity, antigen specificity or one ormore functional properties. The contents of each positive chamber arethen recovered. In one embodiment, recovery of each population is with asingle microcapillary and the chamber contents are pooled in themicrocapillary, and reloaded at limiting dilution onto the same device,or a different microfluidic device. In the embodiment shown in FIG. 2,the cells from array one are reloaded in different chambers at a densityof approximately 1 cell per chamber. The cells from the recoveredpopulation(s) are then rescreened for the same extracellular effect, orfor a different extracellular effect. The contents of the positivechambers from the second array are then recovered to identify antibodysequences of interest, e.g., by next generation sequencing and/or PCR.The antibody sequences, in one embodiment, are sequenced and cloned andtherefore, in one embodiment, the methods provided herein allow for thediscovery of novel antibody genes.

In another embodiment, cell populations displaying the extracellulareffect are recovered using an integrated system microfluidic valves thatallow chambers to be individually addressed (e.g., Singhal et al.(2010). Anal. Chem. 82, pp. 8671-8679, incorporated by reference hereinin its entirety). Notably, the present invention is not limited to thetype of extracellular effect assay carried out on the contents of thepositive chambers of “array 1” (FIG. 2). For example, in someembodiments, it is desirable to further assay the contents of thepositive chambers from “Array 1” via a benchtop method, rather than asecond microfluidic array. Benchtop methods include, for example, RT-PCRand next generation sequencing.

With respect to the single cell and multicellular microfluidic assaysdescribed herein, reference is made herein to a “chamber” in which acell population optionally comprising one or more effector cells isassayed for an extracellular effect, for example, a functional effect ora binding effect. However, one of ordinary skill in the art willrecognize that the devices provided herein provide a massively parallelsystem that incorporates tens of thousands of chambers, and that assaysare carried out in parallel in all or substantially all of the chambers,or all of the chambers in a subarray on one device, on a plurality ofindividual cell populations each optionally comprising an effector cellor a plurality of effector cells. Because of the rarity of some effectorcells, not all cell populations will comprise an effector cell whenpresent in a microfluidic chamber. Fluidic architectures to addressmultiple chambers individually or together, such as multiplexers, aredescribed below.

Some of the embodiments described herein provide one or more of thefollowing features:

The ability to load and concentrate a cell population comprising one ormore effector cells into a microfluidic chamber having a small volume toassay the effector cell products, and to co-localize into within thechamber a readout particle (readout bead, readout cell, etc.) used todetect the presence of an individual effector cell product (e.g.,secreted protein) having a desired property.

The ability to maintain the viability and/or growth of a cellpopulation, assisted by the osmotic bath described herein as well as theability to exchange medium around individual cells, at concentrationsthat have been reported previously to result in poor cell survival orgrowth using conventional culture methods or previously describedmicrofluidic devices.

The ability to concentrate effector cell products within a chamber in atime sufficient to measure a desired effector cell product propertyprior to the effector cell becoming unhealthy or outgrowing itsrespective microfluidic chamber.

The ability to selectively exchange medium contents or add detectionreagents to clusters of cells while maintaining populations of cells inthe microfluidic chambers.

The ability to addressably recover a selected cell population using oneor more microfluidic structures, a manual method or a robotic method.

The ability to transfer recovered populations of cells into a secondarylower-throughput screen that enables the analysis of an effector cellproduct from each single cell in the heterogeneous population, or aclone or plurality of clones generated from each single cell in theheterogeneous population.

The ability to directly analyze the aggregate genetic material fromrecovered heterogeneous populations of single cells and then use thisinformation or genetic material to identify the genes associated withthe cells of interest.

In some embodiments, a method of enriching for effector cells exhibitingan extracellular effect from a starting population of cells thatincludes one or more effector cells that exhibit the extracellulareffect is provided. In one embodiment, the method includes retaining thestarting population of cells in a plurality of microfluidic chambers toobtain a plurality of cell subpopulations. An average number of effectorcells per chamber is greater than Y, the total number of cells in thepopulation is greater than X and an expected fraction of effector cellsin the population is 1/X. The cell subpopulations in the microfluidicchambers are subjected to an extracellular effect assay to identify oneor more chambers containing one or more effector cells exhibiting theextracellular effect. Based on the results of the extracellular effectassay, one or more chambers are then identified as comprising one ormore effector cells exhibiting the extracellular effect. The contents ofthe identified chamber(s) are then recovered to provide an enrichedpopulation of cells. The enriched population of cells enriched for theeffector cells has a fraction of effector cells of 1/Y. In a furtherembodiment, 1/X is less than 0.05, or less than 0.01, or less than0.001. The extracellular effect can be one or more of the extracellulareffects described herein. In one embodiment, the starting population ofcells are peripheral blood mononuclear cells (PBMCs) isolated from ananimal that has been immunized or exposed to an antigen. In anotherembodiment, the starting population of cells is a population of B-cellsisolated from an animal that has been immunized or exposed to anantigen. In yet another embodiment, the source of the startingpopulation of cells is whole blood from the animal that has beenimmunized or exposed to an antigen.

As provided herein, in one aspect, the devices and methods of theinvention are used to assay a cell population optionally comprising oneor more effector cells for the presence of an extracellular effect. Inanother aspect, the devices and methods provided herein allow foridentification of a cell population displaying a variation in anextracellular effect compared to other cell populations. In this aspect,a plurality of individual cell populations are retained in separatemicrofluidic chambers, wherein at least one of the individual cellpopulations comprises one or more effector cells and the separatemicrofluidic chambers further comprise a readout particle populationcomprising one or more readout particles. The cell populations areassayed for the presence of the extracellular effect, whereby thereadout particle population or subpopulation thereof provides a readoutof the extracellular effect. A cell population from amongst theplurality can then be identified that exhibits a variation in theextracellular effect, as compared to one or more of the remaining cellpopulations of the plurality. Once a cell population is identified thatdisplays the variation in the extracellular effect, the population isrecovered and may be further assayed at limiting dilution to identifythe cell or cells within the population responsible for theextracellular effect.

Cell populations that can be analyzed herein are not limited to aspecific type. For example, in one embodiment a starting population ofcells partitioned into individual cell populations in microreactors maybe peripheral blood mononuclear cells (PBMCs) isolated from an animalthat has been immunized or exposed to an antigen. The startingpopulation of cells in another embodiment are B-cells isolated from ananimal that has been immunized or exposed to an antigen. The source ofthe starting population of cells may be whole blood from the animal thathas been immunized or exposed to an antigen.

An “effector cell,” as used herein, refers to a cell that has theability to exert an extracellular effect. The extracellular effect is adirect or indirect effect on a readout particle, as described in detailbelow. The extracellular effect is attributable to the effector cell, ora molecule secreted by the effector cell, for example a signalingmolecule, metabolite, an antibody, neurotransmitter, hormone, enzyme,cytokine. In one embodiment, the effector cell is a cell that secretesor displays a protein (e.g., T cell receptor). In embodiments describedherein, the extracellular effect is characterized via the use of areadout particle, e.g., a readout cell or a readout bead, or a readoutparticle population or subpopulation. For example, in one embodimentdescribed herein, the extracellular effect is the agonizing orantagonizing of a cell surface receptor, ion channel or ATP bindingcassette (ABC) transporter, present on a readout cell or readout bead.In one embodiment, the effector cell is an antibody secreting cell(ASC). An ASC, as used herein, refers to any cell type that produces andsecretes an antibody. Plasma cells (also referred to as “plasma Bcells,” “plasmocytes” and “effector B cells”) are terminallydifferentiated, and are one type of ASC. Other ASCs that qualify as“effector cells” for the purposes of the present invention includeplasmablasts, cells generated through the expansion of memory B cells,cell lines that express recombinant monoclonal antibodies, hybridomacell lines. In another embodiment, the effector cell is a cell thatsecretes a protein. Other cell types that qualify as effector cellsinclude T cells (e.g., CD8+ T cell, and CD4+ T cell), hematopoieticcells, cell lines derived from humans and animals, recombinant celllines, e.g., a recombinant cell line engineered to produce antibodies, arecombinant cell line engineered to express a T cell receptor.

Individual cell populations optionally comprising one or more effectorcells are assayed to determine whether the respective cell populationscomprise an effector cell that exhibits an extracellular effect, or avariation in an extracellular effect as compared to another individualcell population or a plurality thereof. As stated above, when cellpopulations are assayed in parallel on one device, not all cellpopulations will comprise an effector cell, and the methods describedherein allow for the identification of a cell population that containsone or more effector cells. Additionally, a cell population comprisingan effector cell need not include multiple effector cells, or be apopulation of only effector cells. Rather, non-effector cells, inembodiments described herein, are included in the population. Thenon-effector cells can be a majority or minority of the population. Aheterogeneous population comprising an effector cell need not includemultiple effector cells. Rather, a heterogeneous cell population isheterogeneous as long as two cells are heterogeneous with respect to oneanother. A cell population can comprise zero effector cells, oneeffector cell or a plurality of effector cells. Similarly, a cellsubpopulation can comprise zero effector cells, one effector cell or aplurality of effector cells.

The extracellular effect in one embodiment is the binding interactionwith an antigen, or a functional effect. For example, in one embodiment,the extracellular effect is agonism or antagonism of a cell surfacereceptor, agonism or antagonism of an ion channel or agonism orantagonism of an ABC transporter, modulation of apoptosis, modulation ofcell proliferation, a change in a morphological appearance of a readoutparticle, a change in localization of a protein within a readoutparticle, expression of a protein by a readout particle, neutralizationof the biological activity of an accessory particle, cell lysis of areadout cell induced by an effector cell, cell apoptosis of a readoutcell induced by the effector cell, cell necrosis of the readout cell,internalization of an antibody by a readout cell, internalization of anaccessory particle by a readout cell, enzyme neutralization by theeffector cell, neutralization of a soluble signaling molecule, or acombination thereof.

The presence and identification of an effector cell that secretes abiomolecule (e.g., antibody) that binds a target of interest (e.g.,antigen) is readily ascertained in embodiments where the effector cellis present in a heterogeneous cell population comprising a plurality ofeffector cells that secrete antibodies that are not specific to thetarget of interest. In one embodiment, this is achieved in an individualmicrofluidic chamber by first capturing in the chamber, all orsubstantially all of the secreted antibodies of the population on areadout particle(s) (e.g., bead) functionalized to capture antibodies(for example, functionalized with protein G or protein A), addition offluorescently labeled antigen into the chamber and imaging of theparticle(s) to detect the presence or absence of an increase influorescence due to binding of the antigen to immobilized antibody(ies).An estimate of minimum number of antibodies captured on a bead that isrequired for reliable detection may be obtained by performingexperiments to measure antibody secretion from single cells. In oneembodiment, it is possible to detect antigen-specific antibodiessecreted from a single ASC in a heterogeneous population ofapproximately 500 cells. In the case of the present invention, a cellpopulation present in an individual microfluidic chamber can comprisefrom about two to about 500 cells, for example, about two to about 250ASCs. As stated above, a cell population can contain cells other thaneffector cells and not all cell populations will contain an effectorcell. This is particularly true when conventional enrichment protocols(e.g., FACS) are not able to be used to obtain a substantially pure cellpopulation of the same cell type.

With respect to a heterogeneous cell population, all of the cells not beheterogeneous with respect to each other, provided that there are atleast two cells in the population that are heterogeneous with respect toeach other, for example, an effector cell and a non-effector cell. Aheterogeneous cell population may consist of as few as two cells. A cellpopulation or cell subpopulation may consist of a single cell. Inprinciple, a heterogeneous cell population may include any number ofcells that can be maintained in a viable state for the required durationof the extracellular effect assay, e.g., one of the extracellular effectassays provided herein. In one embodiment, the number of cells in a cellpopulation is from 1 cell to about 500 cells per chamber. In oneembodiment, where the imaging of individual cells or readout particlesis required, the number of cells in a population is chosen to beinsufficient to cover the floor of the chamber, so that the cells beingimaged are arranged in a monolayer. Alternatively, the cell populationincludes a number of cells that is insufficient to form a bilayercovering a surface of the chamber.

In some embodiments, larger populations of cells can be present in apopulation within a single microfluidic chamber or microreactor, withoutinhibiting the detection of an effect that stems from a single effectorcell or a small number of effector cells within the particularpopulation. For example, in one embodiment, the number of cells in acell population is from two to about 900, or from about 10 to about 900,or from about 100 to about 900. In another embodiment, the number ofcells in a cell population is from two to about 800, or from about 10 toabout 800, or from about 100 to about 800. In another embodiment, thenumber of cells in a cell population is from two to about 700, or fromabout 10 to about 700, or from about 100 to about 700. In anotherembodiment, the number of cells in a cell population is from two toabout 600, or from about 10 to about 600, or from about 100 to about600. In another embodiment, the number of cells in a cell population isfrom two to about 500, or from about 10 to about 500, or from about 100to about 500. In another embodiment, the number of cells in a cellpopulation is from two to about 400, or from about 10 to about 400, orfrom about 100 to about 400. In another embodiment, the number of cellsin a cell population is from two to about 300, or from about 10 to about300, or from about 100 to about 300. In another embodiment, the numberof cells in a cell population is from two to about 200, or from about 10to about 200, or from about 100 to about 200. In another embodiment, thenumber of cells in a cell population is from two to about 100, or fromabout 10 to about 100, for from about 50 to about 100. In anotherembodiment, the number of cells in a cell population is from two toabout 90, or from about 10 to about 90, or from about 50 to about 900.In yet another embodiment, the number of cells in a cell population isfrom two to about 80, or from 10 to about 80, or from two to about 70,or from about 10 to about 70, or from about two to about 60, or fromabout 10 to about 60, or from about two to about 50, or from about 10 toabout 50, or from about two to about 40, or from about 10 to about 40,or from two to about 30, or from about 10 to about 20, or from two toabout 10. In some embodiments, the majority of cells in a cellpopulation are effector cells.

In one aspect of the invention, a cell or cell population analyzed bythe methods provided herein comprises one or more effector cells, e.g.,an antibody secreting cell (ASC) or a plurality of ASCs. Cells, in oneembodiment, are separated into a plurality of cell populations inthousands of microfluidic chambers, and individual cell populationscomprising one or more effector cells (i.e., within single microfluidicchambers) are assayed for an extracellular effect. One or moreindividual cell populations are identified and recovered if an effectorcell within the one or more populations exhibits the extracellulareffect or a variation in an extracellular effect. The extracellulareffect is determined by the user and in one embodiment, is a bindinginteraction with an antigen, cell surface receptor, ABC transporter oran ion channel.

Although the methods provided herein can be used to identify a singleeffector cell (alone or within a heterogeneous population) based on abinding interaction, e.g., antigen affinity and specificity, theinvention is not limited thereto. Rather, identification of a cellpopulation, in one embodiment, is carried out via the implementation ofa direct functional assay. Accordingly, one aspect of the inventionincludes methods and devices that enable the direct discovery of an ASCwithin a cell population that secretes a “functional antibody,” withoutthe need to initially screen the “functional antibody” for bindingproperties such as affinity and selectivity to an antigen target.

Along these lines, in one aspect, functional antibodies and receptorsdiscoverable by the methods herein are provided. In one embodiment ofthis aspect, the nucleic acid of an effector cell responsible for anextracellular effect is amplified and sequenced. The nucleic acid is agene encoding for a secreted biomolecule (e.g., antibody, or fragmentthereof), or a gene encoding a cell receptor or fragment thereof, forexample a T-cell receptor. The antibody or fragment thereof or cellreceptor or fragment thereof can be cloned and/or sequenced by methodsknown in the art. For example, in one embodiment, an ASC that secretes afunctional antibody discoverable by the methods and devices providedherein is one that modulates cell signaling by binding to a targetedcell surface protein, such as an ion-channel receptor, ABC transporter,a G-protein coupled receptor (GPCR), a receptor tyrosine kinase (RTK) ora receptor with intrinsic enzymatic activity such as intrinsic guanylatecyclase activity.

In one aspect of the invention, a cell population comprising one or aplurality of effector cells is identified in a microreactor, e.g.,microfluidic chamber, based on the result of an extracellular effectassay carried out in the chamber. If an extracellular effect orvariation in extracellular effect is measured in the microreactor, thecell population is recovered and analyzed to determine the effector cellor effector cells within the population responsible for the effect (see,e.g., FIG. 2). In embodiments where the effector cell secretesantibodies, the DNA sequence that encodes the antibody produced by theASC or ASCs can then be determined and subsequently cloned. In oneembodiment, the antibody DNA sequences are cloned and expressed in celllines to provide an immortal source of monoclonal antibody for furthervalidation and pre-clinical testing.

As described herein, a heterogeneous cell population typically includespopulations of cells having numbers ranging from 2 to about 1000, orfrom 2 to about 500, or from about 2 to about 250, or from about 2 toabout 100. A heterogeneous cell population describes a population ofcells that contains at least two cells with a fundamental difference ingenotype, protein expression, mRNA expression or differentiation statewhere at least one of the cells is an effector cell. In particular, aheterogeneous cell population, in one embodiment, include two or moreeffector cells (e.g., from about to about 250 cells) that contain orexpress different immunoglobulin genes, that contain or expressdifferent genes derived from immunoglobin genes, that contain or expressdifferent genes derived from T cell receptor genes, secrete differentimmunoglobulin proteins, secrete different proteins derived fromimmunoglobulin proteins, secrete different proteins or express differentproteins derived from T cell receptor proteins.

In one embodiment, a cell population comprises a population of cellsgenetically engineered to express libraries of molecules that may bind atarget epitope, cells genetically engineered to express genes orfragments of genes derived from cDNA libraries of interest, cellsgenetically engineered with reporters for various biological functions,and cells derived from immortalized lines or primary sources. Notably,clones originating from a single cell, in one embodiment, areheterogeneous with respect to one another due to for example, genesilencing, differentiation, altered gene expression, changes inmorphology, etc. Additionally, cells derived from immortalized lines orprimary sources are not identical clones of a single cell and areconsidered heterogeneous with respect to one another. Rather, a clonalpopulation of cells has originated from a single cell and has not beenmodified genetically, transduced with RNA, transduced with DNA, infectedwith viruses, differentiated, or otherwise manipulated to make the cellsdifferent in a significant functional or molecular way. Cells derivedfrom a single cell, but which naturally undergo somatic hypermutation orare engineered to undergo somatic hypermutation (e.g., by inducingexpression of activation-induced cytidine deaminase, etc.), are notconsidered clones and therefore these cells, when present together, areconsidered a heterogeneous cell population.

As provided throughout, in one aspect, methods and apparatuses areprovided for assaying a plurality of individual cell populations eachoptionally comprising one or more effector cells, in order identify oneor more of the cell populations that includes at least one effector cellhaving an extracellular effect on a readout particle population orsubpopulation thereof, e.g., secretion of a biomolecule having a desiredproperty. Once the cell population(s) is identified, in one embodiment,it is selectively recovered to obtain a recovered cell population. Ifmultiple cell populations are identified, in one embodiment, they arerecovered and pooled, to obtain a recovered cell population. Therecovered cell population is enriched for effector cells, compared tothe starting population of cells originally loaded onto the device inthat the former has a larger percentage of effector cells as compared tothe latter.

Subpopulations of the recovered cell population are assayed for thepresence of a second extracellular effect on a readout particlepopulation, wherein the readout particle population or subpopulationthereof provides a readout of the second extracellular effect. Theextracellular effect can be the same effect that was assayed, on theidentified cell population, or a different extracellular effect. In afurther embodiment, the subpopulations of the identified population eachcomprise from about 1 to about 10 cells. In even a further embodiment,the subpopulations of the identified population comprise an average of 1cell each. One or more of the subpopulations displaying theextracellular effect are then identified and recovered to obtain arecovered subpopulation, which in one embodiment, is enriched foreffector cells. If multiple cell subpopulations are identified, in oneembodiment, they are recovered and pooled, to obtain a recovered cellsubpopulation. The genetic information from the recovered cellsubpopulation can then be isolated, amplified and/or sequenced.

The present invention is not limited by the type of effector cell orcell population that can be assayed according to the methods of thepresent invention. Examples of types of effector cells for use with thepresent invention are provided above, and include primary antibodysecreting cells from any species (e.g., human, mouse, rabbit, etc.),primary memory cells (e.g., can assay IgG, IgM, IgD or otherimmunoglobins displayed on surface of cells or expand/differentiate intoplasma cells), T-cells, hybridoma fusions either after a selection ordirectly after fusion, a cell line that has been transfected (stable ortransient) with one or more libraries of monoclonal antibodies (mAbs)(e.g., for affinity maturation of an identified mAb using libraries ofmutants in fab regions or effector function optimization usingidentified mAbs with mutations in Fc regions or cell lines transfectedwith heavy chain (HC) and light chain (LC) combinations from amplifiedHC/LC variable regions obtained from a person/animal/library orcombinations of cells expressing mAbs (either characterized oruncharacterized to look for synergistic effects, etc.).

Plasma cells (also referred to as “plasma B cells,” “plasmocytes” and“effector B cells”) are terminally differentiated, and are one type ofeffector cell (ASC) that can be assayed with the devices and methods ofthe invention. Other ASCs that qualify as “effector cells” for thepurposes of the present invention include plasmablasts, cells generatedthrough the expansion of memory B cells, cell lines that expressrecombinant monoclonal antibodies, primary hematopoietic cells thatsecrete cytokines, T cells (e.g., CD4+ and CD8+ T-cells), dendriticcells that display protein or peptides on their surface, recombinantcell lines that secrete proteins, hybridoma cell lines, a recombinantcell engineered to produce antibodies, a recombinant cell engineered toexpress a T cell receptor.

It will be appreciated that the cell populations for use with theinvention are not limited by source, rather, they may be derived fromany animal, including human or other mammal, or alternatively, from invitro tissue culture. Cells may be analyzed directly, for example,analyzed directly after harvesting from a source, or after enrichment ofa population having a desired property (such as the secretion ofantibodies that bind a specific antigen) by use of various protocolsthat are known in the art, e.g., flow cytometry. Prior to harvestingfrom an animal source, in one embodiment, the animal is subject to oneor more immunizations. In one embodiment, flow cytometry is used toenrich for effector cells prior to loading onto one of the devicesprovided herein, and the flow cytometry is fluorescence activated cellsorting (FACS). Where a starting cell population is used that has beenenriched for effector cells, e.g., ASCs, and retained as individual cellpopulations in individual microfluidic chambers, the individual cellpopulations need not be comprised entirely of effector cells. Rather,other cell types may be present as a majority or minority. Additionally,one or more of the individual cell populations may contain zero effectorcells.

There are several methods for the enrichment of ASCs derived fromanimals known to those of skill in the art, which can be used to enricha starting population of cells for analysis by the methods and devicesprovided herein. For example, in one embodiment, FACS is used to enrichfor human ASCs using surface markers CD19⁺CD20^(low)CD27^(hi)CD38^(hi)(Smith et al. (2009). Nature Protocols 4, pp. 372-384, incorporated byreference herein in its entirety). In another embodiment, a cellpopulation is enriched by magnetic immunocapture based positive ornegative selection of cells displaying surface markers. In anotherembodiment, a plaque assay (Jerne et al. (1963). Science 140, p. 405,incorporated by reference herein in its entirety), ELISPOT assay(Czerkinsky et al. (1983). J. Immunol. Methods 65, pp. 109-121,incorporated by reference herein in its entirety), droplet assay (Powelet al. (1990). Bio/Technology 8, pp. 333-337, incorporated by referenceherein in its entirety), cell surface fluorescent-linked immunosorbentassay (Yoshimoto et al. (2013), Scientific Reports, 3, 1191,incorporated by reference herein in its entirety) or a cell surfaceaffinity matrix assay (Manz et al. (1995). Proc. Natl. Acad. Sci. U.S.A.92, pp. 1921-1925, incorporated by reference herein in its entirety) isused to enrich for ASCs prior to performing one of the methods providedherein or prior to loading a starting cell population onto one of thedevices provided herein.

In various embodiments, two or more effector cells within a cellpopulation produce and secrete cell products, e.g., antibodies that havea direct or indirect effect on a readout particle population orsubpopulation thereof. With respect to the devices provided herein, itis noted that not all chambers on the device necessarily include a cellpopulation and/or a readout particle population, e.g., empty chambers orpartially filled chambers may be present. Additionally, as providedthroughout, within an individual chamber it may be only a subset of thecells within the cell population, or an individual cell within apopulation that produces and secretes antibodies. In some embodiments,cell populations in the microfluidic chambers do not comprise aneffector cell. These chambers are identifiable by running one or moreextracellular effect assays on each of the cell populations.

In some embodiments, it is desirable to have one or more accessoryparticles, which can include one or more accessory cells present in themicroreactors, e.g., microfluidic chambers, to support the viabilityand/or function of one or more cells in the cell populations or toimplement an extracellular effect assay. For example, in one embodimentan accessory cell, or plurality of accessory cells comprise a fibroblastcell, natural killer (NK) cell, killer T cell, antigen presenting cell,dendritic cell, recombinant cell, or a combination thereof.

An accessory particle or cells, or a population comprising the same, inone embodiment, is delivered to microreactors, e.g., microfluidicchambers together with the cell population. In other words, accessorycells in one embodiment are part of cell populations delivered tomicrofluidic chambers. Alternatively or additionally, the accessoryparticle(s) or accessory cell(s) are delivered to a chamber prior to, orafter, the loading of the heterogeneous population of cells comprisingan effector cell or plurality of effector cells to the microreactor orplurality of microreactors (e.g., microfluidic chamber or plurality ofmicrofluidic chambers).

“Accessory particle” as referred to herein means any particle, includingbut not limited to a protein, protein fragment, cell, that (i) supportsthe viability and/or function of an effector cell, (ii) facilitates anextracellular effect, (iii) facilitates the measurement of anextracellular effect, or (iv) detection of an extracellular effect of aneffector cell.

Accessory particles include but are not limited to proteins, peptides,growth factors, cytokines, neurotransmitters, lipids, phospholipids,carbohydrates, metabolites, signaling molecules, amino acids,monoamines, glycoproteins, hormones, virus particles or a combinationthereof. In one embodiment, one or more accessory particles comprisessphingosine-1-phosphate, lysophosphatidic acid or a combination thereof.

As an example of an accessory cell, in one embodiment, a population offibroblast cells (that do not secrete antibodies) is included within acell population enriched for effector cells (e.g., ASCs) in order toenhance the viability of the effector cell(s) (e.g., ASC(s)) within thepopulation. In another embodiment, a population of NK cells may be addedas accessory particles to implement an antibody-dependent cell-mediatedcytotoxicity assay, where the NK cells will attack and lyse the targetcells upon binding of an antibody on their surface. In embodiments wherefunctional cellular assays are carried out on one or more cellpopulations, it will be appreciated that the effector cell(s) within theone or more cell populations will need to stay viable for an extendedperiod of time while within a chamber of the microfluidic device. Tothis end, accessory particles and/or accessory cells, in one embodiment,are used to sustain the viability of the cell population that optionallycomprises one or more effector cells. As explained below, accessoryparticles, e.g., accessory cells can also be used to sustain or enhancethe viability of a readout cell population or subpopulation thereof,either of which can be a single readout cell.

One advantage of embodiments described herein is that the analysis ofmore than one effector cell within a single microreactor (e.g.,microfluidic chamber), and/or the analysis of single or a few effectorcells in the presence of other cells, allows for much greater assaythroughput and hence the identification and selection of desiredeffector cells that would otherwise be too rare to detect efficiently.This is advantageous in many instances where there are limited methodsto enrich for a desired cell type or where such enrichment hasdeleterious effects such as the reduction of viability of the cellsbeing assayed. One embodiment of the invention that has been builtfeatures an array of 3500 cell analysis chambers. When this device isoperated with an average of 30 cells per chamber, the total assaythroughput is 105,000 cells per experiment. This throughput may be usedfor the selection of tens or hundreds of effector cells that are presentat less than, for example, 1% of the total cell population.

For example, antibody secreting cells may be identified and isolatedwithout the need for enrichment based on surface markers. In B-cellsisolated from peripheral mononuclear blood cells (PBMCs) followingimmunization, the frequency of ASCs may be between 0.01% and 1%. At athroughput of 105,000 cells per device run, it is possible to directlyselect for hundreds of ASCs without further purification. This isparticularly important, since FACS purification of ASCs can reduce cellviability. This is also important because appropriate reagents for theenrichment of ASCs may not be available for a host species of interest.Indeed, following immunization, the frequency of antibody secretingcells in peripheral blood mononuclear cells (PBMCs) may be between 0.01and 1% and is thus detectable by using the microfluidic arrays providedherein, for example, a microfluidic array of 3500 chambers loaded at anaverage density of 30 cells per chamber. Thus, since the isolation ofperipheral blood mononuclear cells (PBMCs) may be performed on anyspecies without specific capture reagents, some of the present methodsprovide for the rapid and economical selection of cells secretingantibodies of interest from any species.

ASCs from basal levels in humans, in one embodiment, are identified bythe methods and devices provided herein. While animals can be immunizedto generate new antibodies against most antigens, the same procedurecannot be performed widely in humans except for approved vaccines.However, humans that have been naturally exposed to an antigen, orvaccinated at some point in their lifespan, typically possess low basallevels of antibody-secreting cells for the antigen. The presentinvention can be used to identify and isolate extremely rare effectorcells secreting specific antibodies from a large number of cells (e.g.,greater than 100,000 per device run). Such methods are used herein forthe discovery of functional antibodies, e.g., as therapeutics forautoimmune diseases and cancers where autoantibodies may be present.

As provided throughout, the present invention relates in part toextracellular effect carried out in a massively parallel fashion insingle microfluidic chambers. The assays are carried out to measure anddetect an extracellular effect exerted by an effector cell, or pluralitythereof, present in a cell population. A population of readout particlesor subpopulation thereof provides a readout of the extracellular effect.For example, the methods described herein allow for the identificationof a heterogeneous cell population which contains an effector cell thatexerts the extracellular effect, e.g., secretion of an antibody specificto a desired antigen, in a background of up to about 250 cells that donot exert the extracellular effect.

“Readout particle,” as used herein, means any particle, including a beador cell, e.g., a functionalized bead or a cell that reports afunctionality or property, or is used in an assay to determine anextracellular effect (e.g., functionality or property) of an effectorcell, or a product of an effector cell such as an antibody. As describedherein, a “readout particle” can be present as a single readout particleor within a homogeneous or heterogeneous population of readout particleswithin a single microfluidic chamber. In one embodiment, a readoutparticle is a bead functionalized to bind one or more biomoleculessecreted by an effector cell (e.g., one or more antibodies), or releasedby an effector or accessory cell upon lysis. A single readout particlemay be functionalized to capture one or more different types ofbiomolecules, for instance a protein and/or nucleic acid, or one or moredifferent monoclonal antibodies. In one embodiment, the readout particleis a bead or a cell that is capable of binding antibodies produced by aneffector cell that produces and/or secretes antibodies. In someembodiments, an effector cell may also be a readout particle, e.g.,where a secretion product of one effector cell in a population has aneffect on a larger, or different, sub-population of the effector cellsor, alternatively, where the secretion product of one effector cell iscaptured on the same cell for readout of the capture.

A “readout cell,” as used herein, is a type of readout particle thatexhibits a response in the presence of a single effector cell or a cellpopulation comprising one or more effector cells, for example one ormore effector cells that secrete antibodies. In various embodiments, thereadout cell is a cell that displays a surface antigen or a receptor(e.g., GPCR or RTK) specific to a secreted molecule. In one embodiment,the binding of the secreted molecule to a readout cell is theextracellular effect. The readout cell may be fluorescently labeledand/or possess fluorescent reporters that are activated upon binding.

As described above, in some embodiments, a cell population subjected tothe methods described herein comprises an ASC or a plurality of ASCs,and the readout particle population or subpopulation thereof displays atarget epitope or a plurality of target epitopes. The readout particlepopulation in one embodiment is a population of beads functionalized tocapture antibodies by a particular epitope or epitopes. Alternatively oradditionally, the readout particle population is specific for anantibody's Fc region, and therefore, does not discriminate betweenantibodies having different epitopes. The readout particle population orsubpopulation thereof, in one embodiment is labeled with afluorescently-conjugated molecule containing the target epitope, forexample to perform an ELISA assay. Fluorescent based antibody andcytokine bead assays are known in the art, see, e.g., Singhal et al.(2010). Anal. Chem. 82, pp. 8671-8679, Luminex® Assays (LifeTechnologies), BD™ Cytometric Bead Array, the disclosures of which areincorporated by reference in their entireties. These methods can be usedherein to determine whether an effector cell has an extracellular effecton a readout particle.

Moreover, as described herein, individual microreactors (e.g.,microfluidic chambers) are structured so that reagent exchange withinthe individual chambers is possible, whereby cross-contamination iseliminated or substantially eliminated between chambers. This allows forthe detection of multiple extracellular effects in a single chamber, forexample, multiple antigen binding effects and/or functional effects in asingle chamber, for example, by exchanging antigens and secondaryantibodies to label the respective binding complexes, followed byimaging. In these serial detection embodiments, the assays can becarried out with the same fluorophores, as each reaction is performedserially after a wash step. Alternatively, different fluorophores can beused to detect different extracellular effects in a serial manner, or inparallel, in one microreactor (e.g., microfluidic chamber).

In another embodiment, the readout particle population is a readout cellpopulation wherein at least some of the readout cells display a targetepitope on their surfaces. In one embodiment, the readout cellpopulation, or a subpopulation thereof, is alive and viable. In anotherembodiment, the readout cell population or a subpopulation thereof isfixed. As will be recognized from the discussion above, where antibodybinding is assayed for, “antibody binding” is considered theextracellular effect of an effector cell or plurality of effector cells.Antibody binding can be detected by, for example, staining of the cellwith one or more fluorescently labeled secondary antibodies. In anotherembodiment, binding of an antibody to the target epitope on a readoutparticle or readout cell causes the death of a readout cell, or someother readout cell response as discussed herein (e.g., secretion ofbiomolecule, activation or inhibition of a cell signaling pathway).

Readout cells in a population may be distinguished, e.g., by featuressuch as morphology, size, surface adhereance, motility, fluorescentresponse. For example, in one embodiment, a population of readout cellsis labeled on their surfaces, or intracellulary, in order to determinewhether the readout cells exhibit a response as chosen by the user ofassay. For example, a calcein, carboxyfluorescein succinymyl esterreporter (CFSE), GFP/YFP/RFP reporters can be used to label one or morereporter cells, including extracellular receptors and intracellularproteins and other biomolecules.

In some embodiments, the readout particle population is a heterogeneousreadout particle population, which can be a heterogeneous readout cellpopulation. Where, for example, an ASC or plurality of ASCs are presentin a cell population, the individual readout particles in the populationmay display different target epitopes, or display two different cellreceptors (e.g., a GPCR or RTK or a combination thereof). Accordingly,the specificity of the extracellular effect, e.g., the specificity of anantibody for a target epitope, or the inhibition of a specific cellsurface receptor, can be assessed. In another embodiment, an effectorcell within a cell population is an ASC, and the readout particlepopulation comprises a heterogeneous bead population thatnon-selectively capture all antibodies (e.g., Fc region specific) and abead population that is specific for a unique target epitope.

In one embodiment, accessory particles are provided to facilitate themeasurement of an extracellular effect, or to facilitate the readout ofan extracellular effect. As described throughout, an extracellulareffect includes an effect that is exhibited by an effector cellsecretion product (e.g., antibody). For example, in one embodiment, anatural killer (NK) cell is provided as an accessory particle, tofacilitate the measurement of lysis of a readout cell. In thisembodiment, the extracellular effect includes lysis of a readout cellthat binds to a specific epitope or cell receptor, by the natural killer(NK) cell, when an antibody secreted by an effector cell binds to theaforementioned readout cell.

In some embodiments, accessory particles include proteins, proteinfragments, peptides, growth factors, cytokines, neurotransmitters (e.g.,neuromodulators or neuropeptides), lipids, phospholipids, amino acids,monoamines, glycoproteins, hormones, virus particles, or a factorrequired to activate the complement pathway, upon binding of an effectorcell secretion product to a readout cell or a combination thereof. Inone embodiment, one or more accessory particles sphingosine-1-phosphate,lysophosphatidic acid or a combination thereof. Various extracellulareffects that are measurable with the devices and methods providedherein, including lysis of the readout cell that binds the antibody, arediscussed in detail below.

For example, cytokines that can be used as accessory particles includechemokines, interferons, interleukins, lymphokines, tumor necrosisfactors. In some embodiments the accessory particles are produced byreadout cells. In some embodiments, a cytokine is used as an accessoryparticle and is one or more of the cytokines provided in Table 1, below.In another embodiment, one or more of the following cytokines is used asan accessory particle: interleukin (IL)-1a, IL-1(3, IL-1RA, IL18, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL17, IL-18, IL-19, IL-20, granulocytecolony-stimulating factor (G-CSF), granulocyte macrophage-colonystimulating factor (GM-CSF), leukemia inhibitor factor, oncostatin M,interferon (IFN)-α, IFN-β, IFN-γ, CD154, lymphotoxin beta (LTB), tumornecrosis factor (TNF)-α, TNF-β, transforming growth factor (TGF)-β,erythropoietin, megakaryocyte growth and development factor (MGDF),Fms-related tyrosine kinase 3 ligand (Flt-3L), stem cell factor, colonystimulating factor-1 (CSF-1), macrophage stimulating factor, 4-1BBligand, a proliferation-inducing ligand (APRIL), cluster ofdifferentiation 70 (CD70), cluster of differentiation 153 (CD153),cluster of differentiation 178 (CD17)8, glucocorticoid induced TNFreceptor ligand (GITRL), LIGHT (also referred to as TNF ligandsuperfamily member 14, HVEM ligand, CD258), OX40L (also referred to asCD252 and is the ligand for CD134), TALL-1, TNF related apoptosisinducing ligand (TRAIL), tumor necrosis factor weak inducer of apoptosis(TWEAK), TNF-related activation-induced cytokine (TRANCE) or acombination thereof

TABLE 1 Representative cytokines and their receptors. CytokineReceptor(s)(Da) and Name Synonym(s) Amino Acids Chromosome MolecularWeight Form Receptor Location(s) Interleukins IL-1α hematopoietin-1 2712q14 30606 CD121a, CDw121b 2q12, 2q12-q22 IL-1β catabolin 269 2q14 20747CD121a, CDw121b 2q12, 2q12-q22 IL-1RA IL-1 receptor antagonist 1772q14.2 20055 CD121a 2q12 IL-18 interferon-γ inducing 193 11q22.2-q22.322326 IL-18Rα, β 2q12 factor Common g chain (CD132) IL-2 T cell growthfactor 153 4q26-q27 17628 CD25, 122,132 10p15-p14, 22q13.1, Xq13.1 IL-4BSF-1 153 5q31.1 17492 CD124,213a13, 132 16p11.2-12.1, X, Xq13.1 IL-7177 8q12-q13 20186 CD127, 132 5p13, Xq13.1 IL-9 T cell growth factor P40144 5q31.1 15909 IL-9R, CD132 Xq28 or Yq12, Xq13.1 IL-13 P600 132 5q31.114319 CD213a1, 213a2, X, Xq13.1-q28, CD1243, 132 16p11.2-12.1, Xq13.1IL-15 162 4q31 18086 IL-15Ra, CD122, 132 10p14-p14, 22q13.1, Xq13.1Common b chain (CD131) IL-3 multipotential CSF, 152 5q31.1 17233 CD123,CDw131 Xp22.3 or Yp11.3, MCGF 22q13.1 IL-5 BCDF-1 134 5q31.1 15238,homodimer CDw125, 131 3p26-p24, 22q13.1 Also related GM-CSF CSF-2 1445q31.1 16295 CD116, CDw131 Xp22.32 or Yp11.2, 22q13.1 IL-6-like IL-6IFN-β2, BSF-2 212 7p21 23718 CD126, 130 1q21, 5q11 IL-11 AGIF 19919q13.3-13.4 21429 IL-11Ra, CD130 9p13, 5q11 Also related G-CSF CSF-3207 17q11.2-q12 21781 CD114 1p35-p34.3 IL-12 NK cell stimulatory 219/3283p12-p13.2/ 24844/37169 CD212 19p13.1, 1p31.2 factor 5q31.1-q33.1heterodimer LIF leukemia inhibitory factor 202 22q12.1-q12.2 22008 LIFR,CD130 5p13-p12 OSM oncostatin M 252 22q12.1-q12.2 28484 OSMR, CD1305p15.2-5p12 IL-10-like IL-10 CSIF 178 1q31-q32 20517, homodimer CDw21011q23 IL-20 176 2q32.2 20437 IL-20Rα, β ? Others IL-14 HMW-BCGF 498 154759 IL-14R ? IL-16 LCF 631 15q24 66694, homotetramer CD4 12pter-p12IL-17 CTLA-8 155 2q31 17504, homodimer CDw217 22q11.1 Interferons IFN-α189 9p22 21781 CD118 21q22.11 IFN-β 187 9p21 22294 CD118 21q22.11 IFN-γ166 12q14 19348, homodimer CDw119 6q23-q24 TNF CD154 CD40L, TRAP 261Xq26 29273, homotrimer CD40 20q12-q13.2 LT-β 244 6p21.3 25390,heterotrimer LTβR 12p13 TNF-α cachectin 233 6p21.3 25644, homotrimerCD120a, b 12p13.2, 1p36.3-p36.2 TNF-β LT-α 205 6p21.3 22297,heterotrimer CD120a, b 12p13.2, 1p36.3-p36.2 4-1BBL 254 19p13.3 26624,trimer? CDw137 (4-1BB) 1p36 APRIL TALL-2 250 17p13.1 27433, trimer?BCMA, TACI 16p13.1, 17p11.2 CD70 CD27L 193 19p13 21146, trimer? CD2712p13 CD153 CD30L 234 9q33 26017, trimer? CD30 1p36 CD178 FasL 281 1q2331485, trimer? CD95 (Fas) 10q24.1 GITRL 177 1q23 20307, trimer? GITR1p36.3 LIGHT 240 16p11.2 26351, trimer? LTbR, HVEM 12p13, 1p36.3-p36.2OX40L 183 1q25 21050, trimer? OX40 1p36 TALL-1 285 13q32-q34 31222,trimer? BCMA, TACI 16p13.1, 17p11.2 TRAIL Apo2L 281 3q26 32509, trimer?TRAILR1-4 8p21 TWEAK Apo3L 249 17p13.3 27216, trimer? Apo3 1p36.2 TRANCEOPGL 317 13q14 35478, trimer? RANK, OPG 18q22.1, 8q24 TGF-β TGF-β1 TGF-β390 19q13.1 44341, homodimer TGF-βR1 9q22 TGF-β2 414 1q41 47747,homodimer TGF-βR2 3p22 TGF-β3 412 14q24 47328, homodimer TGF-βR31p33-p32 Miscellaneous hematopoietins Epo erythropoietin 193 7q21 21306EpoR 19p13.3-p13.2 Tpo MGDF 353 3q26.3-q27 37822 TpoR 1p34 Flt-3L 23519q13.1 26416 Flt-3 13q12 SCF stem cell factor, c-kit 273 12q22 30898,homodimer CD117 4q11-q12 ligand M-CSF CSF-1 554 1p21-p13 60119,homodimer CD115 5q33-q35 MSP Macrophage stimulating 711 3p21 80379CDw136 3p21.3 factor, MST-1 Adapted from Cytokines, Chemokines and TheirReceptors. Madame Curie Bioscience Database. (Landes Biosceince)

In one embodiment, an accessory particle is a cytokine or other factoroperable to stimulate a response of the readout cell. For example,readout cells may be incubated with an effector cell or pluralitythereof and pulsed with a cytokine that is operable to affect thereadout cell. Alternatively or additionally, cytokine-secreting cellsoperable to affect the readout particles are provided to the chamber asaccessory cells. Neutralization of the secreted cytokines by an effectorcell secretion product in one embodiment, are detected by the absence ofthe expected effect of the cytokine on the readout cell. In anotherembodiment, an accessory particle is provided and is a virus operable toinfect one or more readout cells, and neutralization of the virus isdetected as the reduced infection of readout cells by the virus.

Notably, and as should be evident by the discussion provided aboveregarding accessory particles, the extracellular effect measurable anddetectable by the devices and methods provided herein is not limited tothe binding of an antibody to a target epitope. Rather an extracellulareffect as described herein, in one embodiment, is a functional effect.The functional effect, in one embodiment is apoptosis, modulation ofcell proliferation, a change in a morphological appearance of thereadout particle, a change in aggregation of multiple readout particles,a change in localization of a protein within the readout particle,expression of a protein by the readout particle, secretion of a proteinby the readout particle, triggering of a cell signaling cascade, readoutcell internalization of a molecule secreted by an effector cell,neutralization of an accessory particle operable to affect the readoutparticle.

Once an extracellular effect is identified in a microreactor (e.g.,microfluidic chamber) comprising a cell population, the population isrecovered and a downstream assay can be performed on subpopulations ofthe recovered cell population, to determine which effector cell(s) isresponsible for the measured extracellular effect. The downstream assayin one embodiment is a microfluidic assay. In a further embodiment, thedownstream assay is carried out on the same device as the firstextracellular effect assay. However, in another embodiment, thedownstream assay is carried out in a different microfluidic device, orvia a non-microfluidic method, for example, a benchtop single cellreverse transcriptase (RT)-PCR reaction. Antibody gene sequences ofidentified and recovered effector cells in one embodiment are isolated,cloned and expressed to provide novel functional antibodies.

Although functional effects of single ASCs are measurable by the methodsand devices provided herein, affinity, binding and specificity can alsobe measured as the “effect” of an effector cell, e.g., an effect of aneffector cell secretion product. For example, the binding assay providedby Dierks et al. (2009). Anal. Biochem. 386, pp. 30-35, incorporated byreference herein in its entirety, can be used in the devices providedherein to determine whether an ASC secretes an antibody that binds to aspecific target.

In another embodiment, the extracellular effect is affinity for anantigen or cell receptor, and the method described by Singhal et al.(2010). Anal. Chem. 82, pp. 8671-8679, incorporated by reference hereinfor all purposes, is used to assay the extracellular effect.

In one embodiment, parallel analyses of multiple extracellular effectsare carried out in one microreactor (e.g., microfluidic chamber) byemploying multiple types of readout particles. Alternatively oradditionally, parallel analyses of multiple functional effects arecarried out on a single microfluidic device by employing differentreadout particles in at least two different chambers.

In one embodiment, the readout particle is an enzyme that is present asa soluble molecule, or that is tethered to the microfluidic chambersurface or to another physical support in the readout zone of thechamber. In this case, the binding of an antibody that inhibits theenzymatic activity of the readout particle, in one embodiment, isdetected by reduced signal that reports on the enzymatic activity,including a fluorescent signal or colorometric signal or precipitationreaction.

In various embodiments, determining whether an effector cell or multipleeffector cells within a cell population have an extracellular effect ona population of readout particles or subpopulation thereof involveslight and/or fluorescence microscopy of the microfluidic chambercontaining the cell population. Accordingly, one embodiment of theinvention involves maintaining the readout particle population in asingle plane so as to facilitate imaging of the particles by microscopy.In one embodiment, a readout particle population in a chamber ismaintained in a single plane that is imaged through the device material,or portion thereof (e.g., glass or PDMS) to generate one, or many,high-resolution images of the chamber. In one embodiment, ahigh-resolution image is an image comparable to what is achieved usingstandard microscopy formats with a comparable optical instrument (lensesand objectives, lighting and contrast mechanisms, etc.).

According to one aspect of the invention, a method is provided foridentifying a cell population comprising one or more effector cells thatdisplays a variation in an extracellular effect. In one embodiment, themethod comprises retaining a plurality of individual cell populations inseparate microreactors (e.g., microfluidic chambers), wherein at leastone of the individual cell populations comprises one or more effectorcells and the contents of the separate microfluidic chambers furthercomprise a readout particle population comprising one or more readoutparticles. The cell populations and the readout particle populations areincubated within the microreactor, and the cell populations are assayedfor the presence of the extracellular effect. The readout particlepopulation or a subpopulation thereof provides a direct or indirectreadout of the extracellular effect. Based on the results of the assay,it is determined whether a cell population from amongst the pluralitycomprising one or more effector cells that exhibit the extracellulareffect.

In some embodiments, one or more of the individual cell populations andthe readout particle populations are positioned in an “effector zone”and “readout zone” of a microreactor, respectively. However, theinvention is not limited thereto. When effector zones and readout zonesare employed, in one embodiment, each are essentially defined by thenature of the cells or particles positioned within it. That is, the oneor more effector cells are segregated into the effector zone and the oneor more readout particles are segregated into the readout zone. Theeffector zone is in fluid communication with the readout zone.Accordingly, in some embodiments, where a cell population and readoutparticles are provided to a chamber at low densities, e.g., less thantwo effector cells and readout particles per chamber, physicalseparation of the readout particles from an effector cell isaccomplished. It will be recognized however, that the invention need notbe practiced with discrete zones within a chamber. As should be evidentby the present description, such a separation is not necessary to carryout the extracellular effect assays set forth herein.

The cell population and readout particle population can be loadedsimultaneously into a microreactor (e.g., through different inlet portsor in one mixture through a single inlet port. Alternatively, theeffector cells and readout particles are loaded serially into amicroreactor (e.g., microfluidic chamber). A person skilled in the artwill understand that the cell population can be provided to amicroreactor prior to (or after) the loading of the readout particle(s)to the chamber. However, it is possible that the readout particlepopulation and cell population be provided together as a mixture.

The devices and methods disclosed herein provide robust platforms forcarrying out one or more extracellular effect assays on a plurality ofcell populations, for example, to identify a cell population from theplurality displaying a variation in an extracellular effect. Thevariation is attributable to one or more effector cells present in thecell population. Each cell population is confined to a singlemicrofluidic chamber, and the effector cell assay performed in eachchamber of a device can be the same (e.g., all cell toxicity assays, allbinding assays, etc.) or different (e.g., one binding assay, oneapoptosis assay). The addressability of specific chambers on the device,for example with different readout particles and reagents for performingeffector cell assays, makes diverse analyses methods possible.

The microfluidic devices described herein comprise a plurality ofchambers, and each of the plurality has the capability for housing acell population and a readout particle population, to determine whetherany effector cell(s) in the cell population demonstrate an extracellulareffect on a readout particle population or a subpopulation thereof. Areadout particle population may consist of a single readout particle. Asprovided below, devices provided herein are designed and fabricated toassay a plurality of cell populations on one device, for example, wherehundreds to thousands of cell populations are assayed on one device inorder to identify one or more of the cell populations displaying avariation in the extracellular effect. At least a portion of the cellpopulations are heterogeneous with respect to one another. Thevariation, for example, is a variation compared to the extracellulareffect displayed by other populations of the plurality. For example, inone embodiment, a method is provided for identifying a cellpopulation(s) from among a plurality of cell populations, wherein theselected population(s) has a variation in an extracellular effect, ascompared to the remaining cell populations. The variation in theextracellular effect is detectable for example, by a difference influorescence intensity of one of the chambers compared to the remainingplurality or a subplurality thereof.

According to one aspect of the invention, a method is provided foridentifying a cell population from amongst a plurality of cellpopulations that displays a variation in an extracellular effect,wherein the extracellular effect is induced by the binding of one ormore soluble factors secreted from an effector cell(s) to a readoutparticle population or subpopulation thereof. The readout particlepopulation can be a homogeneous or a heterogeneous population. In oneembodiment, a method provided herein involves retaining individualreadout particle populations within different chambers of a microfluidicdevice. The number of readout particles analyzed can vary and will bedetermined according to considerations analogous to those for cellpopulations as outlined above.

An individual readout particle population and cell population areretained in a single chamber of one of the microfluidic devices providedherein. Optionally, the chamber is substantially isolated from otherchambers of the microfluidic device that also comprise individual cellpopulations and a readout particle population, for example, to minimizecontamination between chambers. However, isolation is not necessary topractice the methods provided herein. In one embodiment, where isolationis desired, isolation comprises fluidic isolation, and fluidic isolationof chambers is achieved by physically sealing them, e.g., by usingvalves surrounding the chambers. However, isolation in anotherembodiment is achieved without physically sealing the chamber, bylimiting fluid communication between chambers so as to precludecontamination between one chamber and another chamber of themicrofluidic device.

Once a chamber or plurality of chambers each comprising a cellpopulation which optionally comprise one or more effector cells, and areadout particle population is isolated, the chamber or chambers, andspecifically the cell population with the chamber (or chambers) isincubated. It will be appreciated that an initial incubation step canoccur prior to the addition of readout particles, and/or after readoutparticles are added to a chamber comprising a cell population.

For example, an incubation step can include a medium exchange to keepthe cell population healthy, or a cell wash step. Incubation can alsocomprise addition of accessory particles used to carry out anextracellular effect assay.

An incubating step, in one embodiment, includes controlling one or moreof properties of the chamber, e.g., humidity, temperature and/or pH tomaintain cell viability (effector cell, accessory cell or readout cell)and/or maintain one or more functional properties of the a cell in thechamber, such as secretion, surface marker expression, gene expression,signaling mechanisms, etc. In one embodiment, an incubation stepincludes flowing a perfusing fluid through the chamber. The perfusingfluid is selected depending on type of effector cell and/or readout cellis in the particular chamber. For example, a perfusing liquid in oneembodiment, is selected to maintain cell viability, e.g., to replenishdepleted oxygen or remove waste productions, or to maintain cellularstate, e.g., to replenish essential cytokines, or to assist in assayingthe desired effect, e.g., to add fluorescent detection reagents.Perfusion can also be used to exchange reagents, for example, to assayfor multiple extracellular effects in a serial manner.

In another embodiment, incubating a cell population includes flowing aperfusing fluid through the chamber to induce a cellular response of areadout particle (e.g., readout cell). For example, the incubating stepin one embodiment comprises adding a fluid comprising signalingcytokines to a chamber comprising the cell population. The incubatingstep can be periodic, continuous, or a combination thereof. For example,flowing a perfusing fluid to a microfluidic chamber or chambers isperiodic or continuous, or a combination thereof. In one embodiment, theflow rate of an incubating fluid (e.g., perfusing fluid) is controlledby integrated microfluidic micro-valves and micro-pumps. In anotherembodiment, the flow of an incubating liquid is pressure driven, forexample by using compressed air, syringe pumps or gravity to modulatethe flow.

Once individual chambers within a device are provided with a cellpopulation and a readout particle population, a method is carried out todetermine whether a cell within the population exhibits an extracellulareffect on the readout particle population or subpopulation thereof. Thecell population and, readout particle population and/or subpopulationthereof, as appropriate, is then examined to determine whether or not acell(s) within a population exhibits the extracellular effect, or ifcompared to other cell populations, a variation in the extracellulareffect. It is not necessary that the specific cell or cells exhibitingthe extracellular effect or variation thereof be identified within thechamber, so long as the presence and/or variation is detected within thechamber. In one embodiment, once a cell population is identified asexhibiting an extracellular effect or variation of the extracellulareffect, the cell population is recovered for further characterization toidentify the specific effector cell(s) responsible for the extracellulareffect or variation thereof. In another embodiment, once the cellpopulation is identified as exhibiting the extracellular effect orvariation in extracellular effect, it is recovered and the nucleic acidfrom the cell population is amplified and sequenced.

The extracellular effect in one embodiment is a binding interactionbetween the protein produced by an effector cell(s) and a readoutparticle, e.g., a bead or a cell. In one embodiment, one or more of theeffector cells in the population is an antibody producing cell, and thereadout particle includes an antigen having a target epitope. Theextracellular effect in one embodiment is the binding of an antibody toan antigen and the variation, for example, is greater binding ascompared to a control chamber or other populations of the plurality.Alternatively, the variation in the extracellular effect is the presenceof an effector cell that secretes an antibody with a modulated affinityfor a particular antigen. That is, the binding interaction is a measureof one or more of antigen-antibody binding specificity, antigen-antibodybinding affinity, and antigen-antibody binding kinetics. Alternativelyor additionally, the extracellular effect is a modulation of apoptosis,modulation of cell proliferation, a change in a morphological appearanceof a readout particle, a change in localization of a protein within areadout particle, expression of a protein by a readout particle,neutralization of the biological activity of an accessory particle, celllysis of a readout cell induced by the effector cell, cell apoptosis ofthe readout cell induced by the effector cell, readout cell necrosis,internalization of an antibody, internalization of an accessoryparticle, enzyme neutralization by the effector cell, neutralization ofa soluble signaling molecule or a combination thereof. In someembodiments, at least two different types of readout particles areprovided to a chamber in which one of the types of readout particlesdoes not include the target epitope.

Different types of readout particles may be distinguished by one or morecharacteristics such as by fluorescence labeling, varying levels offluorescence intensity, morphology, size, surface staining and locationin the chamber.

Once incubated with a cell population comprising an effector cell(s),the readout particle population or subpopulation thereof is examined todetermine whether one or more cells within the cell population exhibitsan extracellular effect on one or more readout particles, whether director indirect, or a variation in the extracellular effect. Cellpopulations are identified that have a variation in the extracellulareffect assayed for, and then recovered for downstream analysis.Importantly, as provided throughout, it is not necessary that thespecific effector cell(s), having the particular extracellular effect onthe one or more readout particles be identified so long as the presenceof the extracellular effect is detected within a particular microreactor(e.g., microfluidic chamber).

In some embodiments, the one or more effector cells within the cellpopulation secretes defined biomolecules, e.g., antibodies, and theextracellular effect of these factors is evaluated on a readout particleor a plurality of readout particles (e.g., readout cells) in order todetect a cell population that demonstrates the extracellular effect. Theextracellular effect, however, is not limited to an effect of a secretedbiomolecule. For example, in one embodiment, the extracellular effect isan effect of a T-cell receptor, for example, binding to an antigen.

In one embodiment, a readout particle population is a heterogeneouspopulation of readout cells and comprises readout cells engineered toexpress a cDNA library, whereby the cDNA library encodes for cellsurface proteins. The binding of antibody to these cells is used torecover, and possibly to analyze, cells that secrete antibodies thatbind to a target epitope.

In some embodiments, methods for measuring an extracellular effect on areadout particle population or subpopulation thereof includes theaddition of one or more accessory particles to the chamber where theeffect is being measured. For example, at least one factor required toactivate complement on binding of an antibody to the readout cells maybe provided as an accessory particle. As provided above, a naturalkiller cell or plurality thereof, in one embodiment, is added to achamber as an accessory cell in cases where cell lysis is beingmeasured. One of skill in the art will be able to determine whataccessory particles are necessary, based on the assay being employed.

In some embodiments, where one or more readout particles include areadout cell displaying or expressing a target antigen, a natural killercell, or a plurality thereof is provided to the chamber as an “accessorycell(s)” that facilitates the functional effect (lysis) being measured.The accessory cell can be provided to the chamber with the cellpopulation, the readout particle(s), prior to the readout particle(s)being loaded, or after the readout particle(s) is loaded into thechamber. In embodiments where a natural killer cell is employed, thenatural killer cell targets one or more readout cells to which anantibody produced by an effector cell has bound. The extracellulareffect may thus include lysis of the one or more readout cells by thenatural killer cell. Lysis can be measured by viability dyes, membraneintegrity dyes, release of fluorescent dyes, enzymatic assays, etc.

In some embodiments, the extracellular effect is neutralization of anaccessory particle (or accessory reagent) operable to affect the readoutparticle, e.g. a cytokine (accessory particle) operable to stimulate aresponse of the at least one readout cell. For example,cytokine-secreting cells operable to affect the readout particle cellsmay further be provided to the chamber. Neutralization of the secretedcytokines by an effector cell may be detected as the absence of theexpected effect of the cytokine on the readout cell, e.g. proliferation.In another embodiment, the accessory particle is a virus operable toinfect the readout cell(s), and neutralization of the virus is detectedas the reduced infection of readout cells by the virus.

In some embodiments, the extracellular effect of one effector cell typemay induce activation of a different type of effector cells (e.g.,secretion of antibodies or cytokine) which can then elicit a response inthe at least one readout cell.

As provided throughout, the methods and devices provided herein are usedto identify an effector cell that exhibits a variation in anextracellular effect on a readout particle. The effector cell can bepresent as a single effector cell in a microfluidic chamber, or in acell population within a single chamber. The extracellular effect, forexample, can be an extracellular effect of a secretion product of theeffector cell. In the case where the effector cell is present in alarger cell population, the extracellular effect is first attributed tothe cell population, and the population is isolated and subpopulationsof the isolated population are analyzed to determine the cellular basisfor the extracellular effect. Subpopulation(s) displaying theextracellular effect can then be isolated and further analyzed atlimiting dilution, for example as single cells, or subjected to nucleicacid analysis. In one embodiment, a subpopulation of an isolated cellpopulation contains a single cell.

In one embodiment, the cell population comprises an ASC that secretes amonoclonal antibody. In one embodiment, a readout bead based assay isused in a method of detecting the presence of an effector cell secretingthe antibody amid a background of one or more additional cells notsecreting the antibody. For example, a bead based assay is employed inone embodiment, in a method of detecting an ASC within a cellpopulation, whose antibody binds a target epitope of interest, in thepresence of one or more additional ASCs that secrete antibodies that donot bind the target epitope of interest.

In another embodiment, the ability of an antibody to bind specificallyto a target cell is assessed. Referring to FIG. 3, the assay includes atleast two readout particles, e.g., readout cells 181 and 186, inaddition to at least one effector cell 182 (ASC). Readout cell 181expresses a known target epitope of interest, i.e., target epitope 183,on its surface (either naturally or through genetic engineering) whilereadout cell 186 does not. The two types of readout cells 181 and 186may be distinguishable from themselves and the effector cell 182 by adistinguishable fluorescent marker, other stain or morphology. Effectorcell 182 secretes antibody 184 in the same chamber as readout cells 181and 186. Antibody 184 secreted by effector cell 182 binds to readoutcell 181 via target epitope 183, but does not bind to readout cell 186.A secondary antibody is used to detect the selective binding of antibody184 to readout cell 181. The microfluidic chamber is then imaged todetermine if antibody 184 binding to the readout cell 181 and/or readoutcell 186 has occurred.

Such an assay may also be used to assess the location of antibodybinding on or inside the readout cell(s) using high resolutionmicroscopy. In this embodiment, the readout particles include differentparticle types (e.g., cell types) or particles/cells prepared indifferent ways (for example, by permeabilization and fixation) to assessbinding specificity and/or localization. For instance, such an assaycould be used to identify antibodies that bind the natural conformationof a target on live cells and the denatured form on fixed cells. Such anassay may alternatively be used to determine the location of an epitopeon a target molecule by first blocking other parts of the molecule withantibodies against known epitopes, with different populations of readoutparticles having different blocked epitopes.

In another embodiment, individual heterogeneous readout particlepopulations, (e.g., a readout cell population comprising malignant andnormal cells) and individual cell populations, wherein at least one ofthe individual cell populations comprises an effector cell, are providedto a plurality of microfluidic chambers (e.g., greater than 1000chambers) of one of the devices provided herein. For example, referringto FIG. 4, binding to one or more malignant readout cells 425 andabsence of binding to healthy readout cells 426 in the population ofreadout cells is used to identify a cell population of interestcontaining one or more effector cells producing an antibody of interest,i.e., effector cell 427 producing antibody 428 specific to one or moreof the malignant cells in the population. The two types of readout cells425 and 426 within a chamber are distinguishable by at least oneproperty, for example, fluorescence labeling, varying levels offluorescence intensity, morphology, size, surface staining and locationin the microfluidic chamber. The cells are then incubated within theindividual chambers and imaged to determine if one or more of thechambers includes a cell population that exerts the extracellulareffect, i.e., an ASC that secretes an antibody that binds to a malignantreadout cell but not a healthy readout cell.

If present within a chamber, the cell population containing the one ormore ASCs that secretes an antibody that binds a malignant readout cell425, but not the healthy readout cells 426, can then be recovered toretrieve the sequences of the antibodies within the chamber, or toperform other downstream assays on the individual cells within thepopulation, for example, an assay to determine which effector cell inthe population has the desired binding property. Accordingly, novelfunctional antibodies are provided that are discovered by one or more ofthe methods described herein. The epitope on the malignant readout cell425 may be known or unknown. In the later case, the epitope for theantibody can be identified by the method described below.

In one embodiment, a single cell type can serve as both an effector celland a readout cell. Referring to FIG. 5, this assay is performed with aheterogeneous subpopulation of cells of a single cell type, i.e.,effector cell 430 and readout cell 431, both functionalized to capture amolecule of interest 432 on their surfaces, for instance usingtetrameric antibodies 433 directed against a surface marker and themolecule of interest 432, or an affinity-matrix on the cell to bindbiotinylated antibodies. Referring to FIG. 6, a tetrameric antibodycomplex consists of an antibody (A) 435 that binds the cells and anantibody (B) 436 that binds antibodies secreted from the cells, whereinantibodies A and B are connected by two antibodies 437 that bind the Fcportion of antibodies A and B. Such tetrameric antibody complexes havebeen described in the art (Lansdorp et al. (1986). European Journal ofImmunology 16, pp. 679-683, incorporated by reference herein in itsentirety for all purposes) and are commercially available (StemcellTechnologies, Vancouver Canada). Using these tetramers, the secretedantibodies are captured and linked onto the surface of the cells, thusmaking the effector cells also function as readout particles. Once boundon the surface of cells these antibodies can be assayed for binding, forinstance by the addition of fluorescently labeled antigen. For example,in the case where one is attempting to identify chambers that containcells that secrete a monoclonal antibody that binds to a specifictarget, the antibodies that are secreted from effector cells can becaptured on the surface of these effector cells, and others in thechamber, using appropriate capture agents. Referring again to FIG. 5, itis thus understood that effector cell 430 can also function as a readoutcell, i.e., that the effector cell secreting a molecule of interest 432may more efficiently capture the molecule of interest than readout cell431.

In one embodiment, an extracellular effect assay is performed inparallel in a plurality of microfluidic chambers with a heterogeneouspopulation of readout particles (e.g., a heterogeneous readout cellpopulation) and a substantially homogeneous population of cells in eachchamber, where the individual effector cells within the substantiallyhomogeneous population each produces the same antibody. In a furtherembodiment, the readout particles are readout cells geneticallyengineered to express a library of proteins or protein fragments inorder to determine the target epitope of the antibody secreted by theeffector cells. Referring to FIG. 7, one embodiment of the assayincludes a plurality of effector cells 190 secreting antibody 191. Theassay further includes a heterogeneous readout cell populationcomprising readout cells 192, 193, 194, and 195 displaying epitopes 196,197, 198, and 199, respectively. Effector cells 190 secrete antibodies191 which diffuse toward readout cells 192, 193, 194, and 195.Antibodies 191 bind to readout cell 194 via target epitope 198, but donot bind to readout cells 192, 193, or 195. A secondary antibody may beused to detect the selective binding of antibodies 191 to readout cell194.

Cell populations that include antibodies 191 that bind readout cell 194(or another epitope) can then be recovered from the device and subjectedto a further assay.

In one embodiment, a functional assay is provided to determine whetheran individual ASC within a cell population activates cell lysis of atarget cell, i.e., activates antibody-dependent cell-mediatedcytotoxicity (ADCC). ADCC is a mechanism of cell-mediated immune defensewhereby an effector cell of the immune system lyses a target cell, whosemembrane-surface antigens have been bound by specific antibodies, i.e.,antibodies secreted by an ASC within a particular microfluidic chamberprovided herein. Classical ADCC is mediated by natural killer (NK)cells. However, macrophages, neutrophils and eosinophils can alsomediate ADCC, and can be provided herein as accessory cells to be usedin an ADCC extracellular effect assay.

One embodiment of an ADCC assay provided herein includes a cellpopulation comprising an effector cell or plurality thereof, a readoutcell population (having an epitope of interest on their surfaces) and NKcells as accessory cells. The assay is run to determine if an ASC fromthe cell population induces the NK cells to attack the target cells andlyse them. Referring to FIG. 8, the illustrated embodiment includes cellpopulation comprising ASCs 200 and 201 that secrete antibodies 202 and203, respectively. The illustrated embodiment further includes aheterogeneous readout cell population comprising readout cells 204 and205 displaying epitopes 206 and 207, respectively. ASCs 200 and 201secrete antibodies 202 and 203 which diffuse toward readout cells 204and 205. Antibodies 202 bind to readout cell 205 via target epitope 207,but do not bind to readout cell 204. Antibodies 203 do not bind toeither of readout cells 204 and 205. NK cell 208 detects that readoutcell 205 has been bound by antibodies 202 and proceeds to kill readoutcell 205, while leaving unbound readout cell 204 alone.

A person skilled in the art will understand that the NK cells may beadded to the chamber during or after the incubation of the effectorcells with the readout cells, provided that they are added to thechamber in a manner that facilitates access to the readout cells. The NKcells may be from a heterogeneous population of accessory cells, forinstance peripheral blood mononuclear cells. The NK cells may be from ananimal- or human-derived cell line, and engineered to increase ADCCactivity. A person skilled in the art will further understand that thisassay could be performed with other hematopoietic cell types capable ofmediating ADCC such as macrophages, eosinophils or neutrophils. In thiscase, macrophages, eosinophils or neutrophils are the accessory cells inthe assay. Cell types capable of mediating ADCC can also be animal- orhuman-derived cell lines engineered to increase ADCC activity or toreport signal upon binding antibodies on target cells. In the latter,the target cells are accessory particles while the cells mediating ADCCare the readout particles.

An ADCC extracellular effect assay can be performed on a single effectorcell, a homogeneous cell population, or a heterogeneous cell populationas depicted in FIG. 8. Similarly, an ADCC assay can be performed with asingle readout cell, a homogeneous readout cell population, or aheterogeneous readout cell population, as depicted in FIG. 8. However,in many instances it is desirable to perform an ADCC assay with aplurality of readout cells to avoid the detection of false positivesresulting from the random death of a readout cell.

Cell lysis, in one embodiment, is quantified by a clonogenic assay, bythe addition of a membrane integrity dye, by the loss of intracellularfluorescent molecules or by the release of intracellular molecules insolution. The released biomolecules are measurable directly in solutionor captured onto readout particles for measurement. In some cases,additional accessory molecules are added, such as a substrate for aredox assay or a substrate for an enzymatic assay. Referring to FIG. 9,for example, a cell population comprising effector cell 500 secretingfirst biomolecule 502 and a second effector cell 501 that does notsecrete first biomolecule 502, is incubated in the presence of aheterogeneous readout particle population, including readout cell 503and readout particle 504, and an accessory particle (e.g., naturalkiller cell 505). Binding of first biomolecule 502 to readout cell 503elicits the recruitment of natural killer cell 505 that causes readoutcell 503 to lyse. Upon cell lysis, second biomolecule 506 is releasedfrom readout cell 503 and captured on readout particle 504, a differenttype of readout particle that is functionalized to capture secondbiomolecules 506, e.g., via molecule 507. Molecule 507, in oneembodiment, is a protein, an antibody, an enzyme, a reactive groupand/or a nucleic acid. Captured second biomolecule 506 can be anymolecule present in readout cell 503 such as a protein, enzyme,carbohydrate or a nucleic acid. Binding of the second biomolecule 506 toreadout particle 504, in one embodiment, is quantified using afluorescence assay, a colorimetric assay, a bioluminescence assay or achemoluminescence assay. The assay is performed directly on readoutparticle 504 or indirectly in the surrounding solution, for example, ifcaptured biomolecule 506 is an enzyme that converts a substrate into aproduct with different optical properties. The assay is carried out inmultiple chambers of one of the devices provided herein to determine ifany of the chambers comprises an effector cell that secretes abiomolecule that induces cell lysis.

ADCC assays are known in the art and components are commerciallyavailable. For example, the Guava Cell Toxicity Kit for Flow Cytometry(Millipore), the ADCC Reporter Bioassay Core Kit (Promega), the ADCCAssay (GenScript), the LIVE/DEAD Cell Mediated Cytotoxicity Kit (LifeTechnologies) and the DELFIA cell toxicity assays can be utilized in thedevices provided herein.

In another embodiment, the extracellular effect assay is acomplement-dependent cytotoxicity (CDC) assay. In one CDC embodiment, amethod is provided for identifying the presence of an ASC (or secretedantibody of an ASC) within a cell population that binds to a readoutcell in the presence of soluble factors necessary and/or sufficient toinduce lysis of the readout cell via the classic complement pathway.Accordingly, the assay is to determine whether an antibody secreted byan ASC stimulates lysis of one or more target cells by the classiccomplement pathway.

A CDC assay includes at least one effector cell and at least one readoutcell, and one CDC embodiment is depicted in FIG. 10. The embodimentincludes a cell population that includes effector cell 210 and effectorcell 211 secreting antibodies 212 and 213, respectively. The illustratedembodiment further includes a heterogeneous readout cell populationcomprising readout cell 214 and readout cell 215 displaying epitopes 216and 217, respectively. Effector cells 210 and 211 secrete antibodies 212and 213 which diffuse toward readout cells 214 and 215. Antibodies 212bind to readout cell 215 via target epitope 217, but do not bind toreadout cell 214. Antibodies 213 do not bind to either of readout cells214 and 215. Enzyme C1 218, an accessory particle, and one of thesoluble factors necessary to induce lysis of cells via the classiccomplement pathway, binds to the complex of readout cell 215 withantibody 212 while leaving unbound readout cell 214 alone. Binding ofenzyme C1 208 to the complex of readout cell 215 with antibody 212triggers the classic complement pathway involving additional solublefactors necessary to induce lysis of cells via the class complementpathway (not shown), leading to the rupture and death of readout cell215.

The soluble factors necessary to induce lysis of the readout cells(i.e., the accessory particles necessary for the assay) are added duringor after the incubation of the effector cells with the readout cells,provided that they are added to the chamber in a manner that facilitatesaccess to the readout cells. CDC assays provided herein can be performedon a single effector cell, a homogeneous effector cell population, or aheterogeneous cell population as depicted in FIG. 8. Similarly, the CDCassay can be performed with a single readout cell, a homogeneous readoutcell population or a heterogeneous readout cell population, as depictedin FIG. 8. However, it is desirable in many instances to perform the CDCassay with a readout cell population to avoid the detection of falsepositives resulting from the random death of a readout cell.

Cell lysis by the complement pathway is quantified according to methodsknown to those of skill in the art. For example, cell lysis isquantified by a clonogenic assay, by the addition of a membraneintegrity dye, by the loss of intracellular fluorescent molecules or bythe release of intracellular molecules in solution. The releasedbiomolecules are measured directly in solution or captured onto readoutparticles. In some cases, additional accessory molecules may be addedsuch as a substrate for a redox assay or a substrate for an enzymaticassay. Referring to FIG. 11, for example, a cell population, includingan effector cell 510 secreting a first biomolecule 512 and a secondeffector cell 511 that does not secrete first biomolecule 512, isincubated in the presence of one or more heterogeneous readoutparticles, e.g., readout cell 513 and readout particle 514, in thepresence of accessory particle 515 (e.g., complement proteins). Bindingof biomolecule 512 to readout cell 513 in the presence of accessoryparticles 515 causes readout cell 513 to lyse. Upon cell lysis, secondbiomolecule 516 is released and captured on a readout particle 514, asecond type of readout particle that is functionalized to capturebiomolecule 516, e.g., via molecules 517. Molecules 517 may be one ormore types of molecule such a protein, an antibody, an enzyme, areactive group and/or a nucleic acid. Captured biomolecule 516 is notlimited to type. Rather, captured biomolecule 516 can be any moleculepresent in readout cell 513 such as protein, enzyme, dye, carbohydrateor nucleic acid. Binding of the second biomolecule 516 to readoutparticle 514 is quantified using a fluorescence assay, a colorimetricassay, a bioluminescence assay or a chemoluminescence assay. It isunderstood that the assay may be performed directly on readout particle514 or indirectly in the surrounding solution, for instance if capturedbiomolecule 516 is an enzyme that converts a substrate into a productwith different optical properties.

In another embodiment, an assay is provided to determine whether aneffector cell, alone or within a cell population modulates cell growth.Specifically, the assay is used to determine whether the effector cellsecretes a biomolecule, e.g., a cytokine or antibody that modulates thegrowth rate of readout cells. Referring to FIG. 12, the illustratedembodiment includes a cell population comprising effector cell 220 andeffector cell 221 secreting biomolecules 222 and 223, respectively. Theillustrated embodiment further includes a homogeneous readout cellpopulation comprising readout cell 224. Effector cells 220 and 221secrete biomolecules 222 and 223 which diffuse toward readout cells 224.Biomolecule 222 binds to readout cell 224 to induce growth of readoutcell 224 (represented by perforated lines), whereas biomolecule 223 donot bind to readout cell 224. Microscopic imaging of the chamber is usedto assess the growth of the readout cells 224 relative to cells in otherchambers which are not exposed to the biomolecules.

A cell growth modulation assay can be performed using a cell populationthat optionally comprises one or more effector cells. As noted above, insome embodiments, not all cell populations will contain effector cellsbecause of their rarity and/or difficulty to be enriched for in astarting population that is initially loaded onto one of the devicesprovided herein. The present invention allows for the identification ofthese rare cells by identifying cell populations that comprise one ormore effector cells.

The cell growth modulation assay can also be performed with a singlereadout cell, or a heterogeneous readout cell population in a singlechamber. However, in many instances, it is desirable to perform the cellgrowth modulation assay with a homogeneous readout cell population topermit a more accurate measurement of growth rate.

The cell growth modulation assay, in one embodiment, is adapted toscreen for cells producing biomolecules that inhibit cell growth. Inanother embodiment, the method is adapted to screen for cells producingmolecules that modulate, i.e., increase or decrease, proliferation ratesof readout cells. Growth rate, in one embodiment, is measured by manualor automated cell count from light microscopy images, total fluorescenceintensity of cell expressing a fluorescence, average fluorescenceintensity of cells labeled with a dilutive dye (e.g. CFSE), nucleistaining or some other method known to those of skill in the art.

Commercially available assay to measure proliferation include thealamarBlue® Cell Viability Assay, the CellTrace™ CFSE Cell ProliferationKit and the CellTrace™ Violet Cell Proliferation Kit (all from LifeTechnologies), each of which can be used with the methods and devicesdescribed herein.

In another embodiment, an apoptosis functional assay is provided toselect a cell population comprising one or more effector cells thatinduces apoptosis of another cell, i.e., a readout cell or an accessorycell. In one embodiment, the method is used to identify the presence ofan effector cell that secretes a biomolecule, e.g., a cytokine or anantibody that induces apoptosis of a readout cell or accessory cell.Referring to FIG. 13, the illustrated embodiment includes a cellpopulation comprising effector cell 230 and effector cell 231 secretingbiomolecule 232 and biomolecule 233, respectively. The illustratedembodiment further includes a homogeneous readout cell populationcomprising readout cell 234. Effector cell 230 and effector cell 231secrete biomolecules 232 and 233, which diffuse toward readout cells234. Biomolecule 232 binds to readout cell 234 and induces apoptosis ofreadout cell 234, whereas biomolecule 233 does not bind to the readoutcell. Microscopic imaging of the chamber, in one embodiment, is used toassess apoptosis using, potentially with the inclusion of stains andother markers of apoptosis that are known in the art (e.g., Annexin 5,terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick endlabeling, mitochondrial membrane potential disruption, etc.). In oneembodiment, cell death rather than apoptosis using commerciallyavailable dyes or kits is measured, for example with propidium iodide(PI), LIVE/DEAD® Viability/Cytotoxicity Kit (Life Technologies) orLIVE/DEAD® Cell-Mediated Cytotoxicity Kit (Life Technologies).

An apoptosis assay, in one embodiment, is performed on a cell populationcomprising a single effector cell, a cell population optionallycomprising one or more effector cells or a cell population comprisingone or more effector cells. In one embodiment, the apoptosis assay isperformed with a single readout cell, or a heterogeneous readout cellpopulation. However, in many instances, it is desirable to perform theapoptosis assay with a homogeneous readout cell population to permit amore accurate assessment of apoptosis.

In another embodiment, the microfluidic devices provided herein are usedto select an effector cell that secretes a biomolecule, e.g., a cytokineor antibody that induces autophagy of a readout cell. One embodiment ofthis method is shown in FIG. 14. Referring to FIG. 14, the illustratedembodiment includes a cell population comprising effector cell 441 andeffector cell 442, wherein effector cell 441 secretes biomolecule 443.The illustrated embodiment further includes a heterogeneous readout cellpopulation including first readout cell 444 displaying a target epitope449 and second type of readout cell 445 lacking the target epitope.Effector cell 441 secretes biomolecules 443, which diffuses toward firsttype of readout cell 444 and second type of readout cell 445.Biomolecule 443 binds to first type of readout cell 444 and inducesautophagy of first type of readout cell 444, whereas biomolecules 443does not bind to the second type of readout cell 445. Microscopicimaging of the chamber, in one embodiment, is used to assess autophagyusing cell lines engineered with autophagy reporters that are known inthe art (e.g., FlowCellect™ GFP-LC3 Reporter Autophagy Assay Kit (U205)(EMD Millipore), Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit (LifeTechnologies)).

In one embodiment, an autophagy assay is performed on a cell populationcomprising a single effector cell, a cell population optionallycomprising one or more effector cells or a cell population comprisingone or more effector cells. In one embodiment, an autophagy assay isperformed with a single readout cell, or a heterogeneous readout cellpopulation, or a homogeneous readout cell population. The assay, in oneembodiment, is performed with a homogeneous readout cell population.

In another embodiment, a method is provided for identifying the presenceof an effector cell or to select an effector cell that secretes abiomolecule, e.g., an antibody, that interferes with the ability of aknown biomolecule, e.g., a cytokine, to induce a readout cell to undergoa response. The response is not limited by type. For example, theresponse in one embodiment is selected from cell death, cellproliferation, expression of a reporter, change in morphology, or someother response selected by the user of the method. One embodiment of themethod is provided in FIG. 15. Referring to FIG. 15, the illustratedembodiment includes a cell population comprising effector cell 240 andeffector cell 241 secreting biomolecules 242 and 243, respectively. Theillustrated embodiment further includes a homogeneous readout cellpopulation comprising readout cell 244. Effector cells 240 and 241secrete antibodies 242 and 243, which diffuse into the medium within thechamber. The chamber is pulsed with cytokines 245, which normally have aknown effect on readout cells 244. Antibodies 242 bind to cytokines 245,and thereby prevent them from binding to readout cells 244. Accordingly,the expected response is not observed, indicating that one of effectorcell 240 and 241 is secreting an antibody capable of neutralizing theability of cytokine 245 to stimulate the readout cells 244 to undergo aresponse.

In one embodiment, a cytokine neutralization assay is used to identifythe presence of an effector cell that produces a biomolecule targeting areceptor for the cytokine, present on the readout cell. In this case,binding of an antibody, e.g., antibody 242 to receptors 246 for cytokine245 on readout cells 244 blocks the interaction of the cytokine and thereceptor, so that no response would be stimulated. The cytokinereceptor, in another embodiment, is “solublized” or “stabilized,” forexample, is a cytokine receptor that has been engineered via theHeptares StaR® platform.

The response to the cytokine, in one embodiment, is ascertained bymicroscopic measurements of the associated signaling as known in the artincluding, but not limited to cell death, cell growth, the expression ofa fluorescent reporter protein, the localization of a cellularcomponent, a change in cellular morphology, motility, chemotaxis, cellaggregation, etc. In one embodiment, the response of chambers witheffector cells are compared to chambers lacking effector cells todetermine whether the response is inhibited. If a response is inhibited,the effector cells within the chamber are harvested for furtheranalysis.

In one embodiment, a cytokine assay is performed within an individualmicroreactor on a cell population comprising a single effector cell, acell population optionally comprising one or more effector cells or acell population comprising one or more effector cells. Of course, themethod can be carried out in parallel in a plurality of microchambers ona plurality of cell populations. In one embodiment, the cytokine assayis performed with a single readout cell, or a heterogeneous readout cellpopulation. In one embodiment, the method is carried out with ahomogeneous readout cell population to permit a more accurate assessmentof stimulation, or rather lack thereof, of the readout cells.

Examples of commercially available cytokine-dependent orcytokine-sensitive cell lines for such assays include, but are notlimited to TF-1, NR6R-3T3, CTLL-2, L929 cells, A549, HUVEC (HumanUmbilical Vein Endothelial Cells), BaF3, BW5147.G.1.4.OUAR.1, (allavailable from ATCC), PathHunter® CHO cells (DiscoveRx) and TANGO cells(Life Technologies). A person skilled in the art will understand thatprimary cells (e.g., lymphocytes, monocytes) may also be used as readoutcells for a cytokine assay.

In one embodiment, a signaling assay is used to identify a cellpopulation comprising one or more effector cells that secretes amolecule (e.g., an antibody or a cytokine) that has agonist activity ona receptor of a readout cell. Upon binding to the receptor, the effecton the readout cell population may include activation of a signalingpathway visualized by expression of a fluorescent reporter,translocation of a fluorescent reporter within a cell, a change ingrowth rate, cell death, a change in morphology, differentiation, achange in the proteins expressed on the surface of the readout cell,etc.

Several engineered reporter cell lines are commercially available andcan be used to implement such an assay. Examples include PathHuntercells® (DiscoverRx), TANGO™ cells (Life Technologies) and EGFP reportercells (ThermoScientific).

In one embodiment, a virus neutralization assay is carried out toidentify and/or select a cell population comprising one or more effectorcells that secretes a biomolecule, e.g., an antibody that interfereswith the ability of a virus to infect a target readout cell or targetaccessory cell. One embodiment of this method is shown in FIG. 16.Referring to FIG. 16, the illustrated embodiment includes a cellpopulation comprising effector cell 250 and effector cell 251 secretingbiomolecules, e.g., antibodies 252 and 253, respectively. Theillustrated embodiment further includes a homogeneous readout cellpopulation comprising readout cell 254. Effector cells 250 and 251secrete biomolecules, e.g., antibodies 252 and 253, which diffuse intothe medium within the chamber. The chamber is then pulsed with virus 255(accessory particle), which normally infects readout cells 254. Antibody252 or 253 binds to virus 255, and thereby prevents the virus frombinding to readout cell 254. Accordingly, the expected infection is notobserved, indicating that one of effector cells 250 or 251 secretes anantibody capable of neutralizing virus 255.

A virus neutralization assay is also amenable for identifying thepresence of an effector cell that produces a biomolecule which binds areceptor for the virus on the readout cell. In this case, binding of anantibody, e.g., antibody 252 to receptor 256 of virus 255 on readoutcell 254 blocks the interaction of the virus and the receptor, so thatno infection would be observed.

Assessment of viral infection may be done using methods known in theart. For example, the virus can be engineered to include fluorescentproteins that are expressed by the readout cell following infection, theexpression of fluorescent proteins within the readout cell that areupregulated during viral infection, the secretion of proteins from areadout cell or accessory cell, which are captured and measured onreadout particles that are increased during viral infection, the deathof the of a readout cell or accessory cell, the change in morphology ofa readout cell or accessory cell, and/or the agglutination of readoutcells.

In one embodiment, within an individual microreactor, a virusneutralization assay is performed on a cell population comprising asingle effector cell, a cell population optionally comprising one ormore effector cells or a cell population comprising one or more effectorcells. In one embodiment, the virus neutralization assay is performedwith a single readout cell, or a heterogeneous readout cell population.In one embodiment, the method is carried out with a homogeneous readoutcell population to permit a more accurate assessment of the stimulation,or rather lack thereof, of the readout cells to undergo the response. Ofcourse, the method can be carried out in parallel in a plurality ofmicrochambers on a plurality of cell populations.

For example, commercially available cell lines for virus neutralizationassays are MDCK cells (ATCC) and CEM-NKR-CCR5 cells (NIH Aids ReagentProgram) can be used with the methods and devices described herein.

In another embodiment, an enzyme neutralization assay is performed todetermine whether an effector cell displays or secretes a biomoleculethat inhibits a target enzyme. One embodiment of the method is providedin FIG. 17. Referring to FIG. 17, the illustrated embodiment includes acell population comprising effector cell 280 and effector cell 281secreting biomolecules, e.g., proteins 282 and 283, respectively. Theillustrated embodiment further includes a homogeneous readout particlepopulation, e.g., beads 284, to which a target enzyme 285 is conjugated.However, in another embodiment, the target enzyme 285 is linked to thesurface of the device, or is soluble. Proteins 282 and 283 diffusethrough the medium and protein 282 binds to target enzyme 285, therebyinhibiting its activity, whereas protein 283 does not bind to the targetenzyme. Detection of the enzymatic activity, or rather lack thereof, ona substrate present in the chamber, in one embodiment, is assessed bymethods known in the art including but not limited to fluorescentreadouts, colorometric readouts, precipitation, etc.

In another embodiment, an enzyme neutralization assay is performed on acell population comprising a single effector cell, a cell populationoptionally comprising one or more effector cells or a cell populationcomprising one or more effector cells, per individual chamber. In oneembodiment, the enzyme neutralization assay is performed with a singlereadout particle in an individual chamber. In one embodiment, an enzymeneutralization assay is carried out on a plurality of cell populationsto identify a cell population having a variation in a response of theassay.

In another embodiment, an assay method is provided for identifying thepresence of an effector cell that displays or secretes a molecule thatelicits the activation of a second type of effector particle, which inturn secretes a molecule that has an effect on a readout particle.Accordingly, in this embodiment, a cell population is provided toindividual microfluidic chambers. One embodiment of this method isprovided in FIG. 18. Referring to FIG. 18, the illustrated embodimentincludes a cell population that includes one effector cell 460 thatdisplays a molecule 461 on its surface (e.g., an antibody, a surfacereceptor, a major histocompatibility complex molecule, etc.), whichactivates an adjacent effector cell of different type, in this caseeffector cell 462, which induces the secretion of another type ofmolecule 463 (e.g., cytokine, antibody) that is captured by the readoutparticle 464. In this example, the readout particles 464 arefunctionalized with an antibody 465 or receptor specific to the secretedmolecule 463.

In another embodiment, the effector cell, upon activation by anaccessory particle, may exhibit changes in phenotype such asproliferation, viability, morphology, motility or differentiation. Inthis case the effector cell is also a readout particle. This effect canbe caused by the accessory particles and/or by autocrine secretion ofproteins by the activated effector cells.

Referring to FIG. 19, the illustrated embodiment includes a cellpopulation comprising effector cell 470 that secretes a molecule 471(e.g., antibody, cytokine, etc.), which activates a second effector cellof different type, in this case effector cell 472. Effector cell 472,once activated, secretes another type of molecule 473 (e.g., cytokine,antibody) that is captured by readout particles 474. In this example,the readout particles 474 are functionalized with an antibody 475 orreceptor specific to the secreted molecule 473.

As provided herein, monoclonal antibodies with low off-rates aredetectable in the presence of a large background (in the same chamber)of monoclonal antibodies that are also specific to the same antigen butwhich have faster off-rates. However, affinity is also measurable withthe devices and methods provided herein, and therefore, on-rate can alsobe measured. These measurements depend on the sensitivity of the opticalsystem as well as the binding capacity of the capture reagents (e.g.,beads). To assay for specificity, the capture reagents (readoutparticles) may be designed to present the epitope of interest so that itonly binds antibodies with a desired specificity.

Referring to FIG. 20, the illustrated embodiment includes a homogeneouscell population secreting antibodies specific for the same antigen butwith different affinities. This assay is used to identify an effectorcell within a population containing at least one effector cell producingan antibody with high affinity. Effector cells 450 and 451 secreteantibodies 453 and 454 with low affinities for a target epitope (notshown) while effector cell 452 secretes antibodies 455 with higheraffinity for the target epitope. Antibodies 453, 454, and 455 arecaptured by a homogeneous readout particle population comprising readoutbead 456. The readout beads are then incubated with a fluorescentlylabeled antigen (not shown), which binds to all antibodies. Upon washingwith a non-labeled antigen (not shown), the fluorescently labeledantigen remains only if readout beads display high-affinity antibody 455on their surfaces.

Referring to FIG. 21, another illustrated embodiment includes effectorcells 260 secreting biomolecules, e.g., antibody 261. The illustratedembodiment further includes a heterogeneous readout particle populationthat optically distinguishable readout particles, e.g., beads 262 and263 displaying different target epitopes 264 and 265, respectively.Antibody 261 diffuses in the chamber, where it binds to epitope 264, butnot 265. Preferential binding of antibody 261 to epitope 264 in oneembodiment, is observed in terms of fluorescence of bead 262 but notbead 263.

In the illustrated embodiment, beads 262 and 263 are opticallydistinguishable by shape in order to assess cross-reactivity. However,readout particles are also be distinguishable by other means, e.g., oneor more characteristics such as fluorescence labeling (includingdifferent fluorescence wavelengths), varying levels of fluorescenceintensity (e.g., using Starfire™ beads with different fluorescenceintensities, morphology, size, surface staining and location in themicrofluidic chamber).

In one embodiment, beads 262 and 263 are optically distinguishable whensegregated into separate readout zones, e.g., by a cell fence.Alternatively, different color fluorophores can be used to opticallydistinguish readout beads.

Alternatively, specificity may be measured by inclusion of anotherantibody that competes with the secreted antibody to bind the targetepitope. For instance, in one embodiment the presence a secretedantibody bound to a readout particle displaying the antigen isidentified with a fluorescently labeled secondary antibody. Thesubsequent addition of a non-labeled competing antibody generated from adifferent host and known to bind a known target epitope on the antigenresults in decreased fluorescence due to displacement of the secretedantibody only if the secreted antibody is bound to the same targetepitope as the competing antibody. Alternatively, specificity ismeasured by adding a mixture of various antigens that compete withbinding of the secreted antibody to the target epitope if the secretedantibody has low specificity. Alternatively, specificity is measured bycapturing secreted antibody on a bead and then using differentiallylabeled antigens to assess the binding properties of the secretedantibody.

The specificity measurements described herein, when conducted on a cellpopulation that includes two or more effector cells that secreteantibody that bind one of the targets, are inherently polyclonalmeasurements.

In various embodiments of the invention, a method for identifying thepresence of an effector cell secreting a biomolecule is coupled with theanalysis of the presence or absence of one or more intracellularcompounds of the effector cell. Referring to FIG. 22, a cell populationcomprising at least one effector cell type 520 that secretes abiomolecule of interest 522 (e.g., an antibody, or a cytokine) andanother effector cell type 521 that does not secrete the biomolecule ofinterest, is incubated in the presence of a readout particle populationcomprising readout particle 523 functionalized to capture thebiomolecule of interest. After an incubation period, the cell populationincluding effector cell types 520 and 521 is lysed to release theintracellular contents of the cells in the population. Readout particles523 are also functionalized to capture an intracellular biomolecule ofinterest 524 (e.g., a nucleic acid, a protein, an antibody, etc.). Celllysis can be achieved by different methods known to those of skill inthe art.

In one embodiment, methods are provided for identifying polyclonalmixtures of secreted biomolecules with desirable binding properties.Assays may be performed as with heterogeneous mixtures of effector cellsproducing antibodies with known affinities for a target epitope, targetmolecule, or target cell type. Binding of the target in the context ofthe mixture can then be compared to binding of the target in the contextof the individual effector cells alone to determine, for example, ifmixtures provide enhanced effects.

Multifunctional analysis that combines the binding and/or functionalassays described herein may be performed by having multiple readoutregions, multiple effector regions, multiple readout particle types, orsome combination thereof. For example, perfusion steps can be carriedout between extracellular effect assays to exchange reagents fordifferent functional experiments.

One embodiment of a multifunctional assay is provided in FIG. 23.Referring to FIG. 23, a microfluidic chamber for simultaneouslyevaluating the extracellular effect of an effector cell on three subsetsof readout particles is shown generally at 410. Microfluidic chamber 410includes cell fences 420 and 421 which divide the chamber into fourzones. While cell fences 420 and 421 are depicted in this example asbeing at right angles to each other, a person skilled in the art willunderstand that the precise positioning of the fence could vary so longas four zones are created. Moreover, a person skilled in the art willunderstand that cell fences are not necessary to carry out amultifunctional assay (either serially or in parallel). In oneembodiment, a structure other than a cell fence is included to result inthe creation zones, so long as the different zones are addressable interms of the delivery of cell populations and readout particles. Inanother embodiment, the multifunctional assay is carried out without acell fence or structure. Rather, each assay is performed in the chambersimultaneously or serially, for example, with fluorescent molecules thatemit light at different wavelengths.

In the embodiment depicted in FIG. 23, effector cells 411 and 422secreting antibodies 419 and 423, respectively, are delivered to theupper left region of chamber 410, thereby defining effector zone 415.Readout particles 412, 413, and 414 are delivered to the remainingregions, thereby defining readout zones 416, 417, and 418, respectively.Readout particles 412, 413, and 414, in one embodiment, make up aheterogeneous population of readout particles. For example, in oneembodiment, readout particles 412 and 414 are beads of different sizesdisplaying different epitopes of the same antigen, and readout particles413 are cells displaying the antigen in the presence of a natural killercell 424. Accordingly, the presence or absence of an effector cell thatcan selectively bind a given epitope and induce the killing of readoutcells by natural killer cells can be evaluated simultaneously in asingle chamber.

Alternatively, readout particles 412, 413, and 414 are identical andallow for multiple independent measurements of an extracellular effectconferred by a single set of effector cells in a single chamber.Alternatively, particles 412 and 414 are distinguishable and the assayis performed serially.

In one embodiment, the presence of one or more extracellular effects isanalyzed. Depending on the effect, one of skill in the art willrecognize that the presence or the absence of the combined extracellulareffects may be desired. Similarly, it is also possible that the desiredproperties include the presence of one or more types of extracellulareffect and the absence of a different type of extracellular effect. Forinstance, in one embodiment, a multifunctional assay is used to identifyan effector cell secreting an antibody that binds to a receptor epitopeon a readout cell but that does not induce the activation of thecorresponding signaling pathway.

In one embodiment, a multifunctional assay is carried out in a chambercomprising multiple effector zones, for example, by introducingdifferent effector cells, or combinations of effector cells, into thedifferent regions. For example, in one embodiment, different cellpopulations producing antibodies of known affinity for a target antigenare introduced into different effector zones of a chamber. Binding ofthe target in the context of the mixtures can then be compared tobinding of the target in the context of the individual effector cellsalone. Accordingly, such use of multiple effector zones allows for thescreening of multiple combinations in a single chamber.

In one embodiment of the assays provided herein, after readout particlesare incubated with a cell population in a microfluidic chamber, afluorescent measurement is taken to determine if a cell within thepopulation demonstrates an extracellular effect. In this embodiment, thereadout particle population is fluorescently labeled and a change influorescence is correlated with the presence and/or size of theextracellular effect. The readout particle population can befluorescently labeled directly or indirectly. In some embodiments, asprovided throughout, accessory particles (e.g., accessory cells) areprovided to a chamber to aid in facilitating the fluorescent readout. Aswill be appreciated by one of skill in the art, care is taken to designassays that provide readout particles and effector cells in one focalplane, to allow for accurate imaging and fluorescent measurement.

In one embodiment, readout particle responses are monitored usingautomated high resolution microscopy. For example, imaging can bemonitored by using a 20× (0.4 N.A.) objective on an Axiovert 200 (Zeiss)or DMIRE2 (Leica) motorized inverted microscope. Using the automatedmicroscopy system provided herein allows for complete imaging of a 4000chamber array, including 1 bright-field and 3 fluorescent channels, inapproximately 30 minutes. This platform can be adapted to various chipdesigns, as described in Lecault et al. (2011). Nature Methods 8, pp.581-586, incorporated by reference herein in its entirety for allpurposes. Importantly, the imaging methods used herein achieve asufficient signal in effect positive chambers while minimizingphotodamage to cells.

In one embodiment, the effector cell assays provided herein benefit fromlong-term cell culture, and therefore require that the effector cellsmaintained in the device are viable and healthy cells. It will beappreciated that in embodiments where readout cells or accessory cellsare used in an effector cell assay, that they too be maintained in ahealthy state and are viable and healthy. The fluidic architecturesprovided herein enable careful and precise control of medium conditionsto maintain effector and readout cell viability so that functionalassays can be carried out. For example, some cell types requireautocrine or paracrine factors that depend on the accumulation ofsecreted products. For instance, CHO cell growth rates are highlydependent on seeding density. Confining a single CHO cell in 4-nLchamber corresponds to a seeding density of 250,000 cells/ml, which iscomparable to conventional macroscale cultures. As shown in FIG. 73,single CHO cells have a higher growth rate in a microfluidic device thanwhen plated in a multiwell plate. Because they thrive at high seedingdensities, CHO cells may not require perfusion for multiple days.However, other cell types, in particular those that arecytokine-dependent (e.g. ND13 cells, BaF3 cells, hematopoietic stemcells), typically do not reach high concentrations in macroscale cultureand may require frequent feeding in the microfluidic device to preventcytokine depletion. The cytokines may be added to the medium or producedby feeder cells. For instance, bone marrow derived stromal cells andeosinophils have been shown to support the survival of plasma cellsbecause of their production of IL-6 and other factors (Wols et al.,(2002), Journal of Immunology 169, pp. 4213-21; Chu et al. (2011),Nature Immunology, 2, pp. 151-159, incorporated by reference herein intheir entireties). In this case the perfusion frequency can be modulatedto allow sufficient accumulation of paracrine factors while preventingnutrient depletion.

In one aspect, the present invention provides a method for determiningwhether a cell population optionally comprising one or more effectorcells exerts an extracellular effect a readout particle (e.g., a cellcomprising a cell surface receptor). The effector cell can be present ina heterogeneous cell population, a homogeneous population, or as asingle cell. In one embodiment, the effector cell is an antibodysecreting cell. The one or more extracellular properties, in oneembodiment, comprise an extracellular effect on a readout particle, forexample, the inhibition (antagonism) or activation (agonism) of a cellsurface receptor (e.g., the agonist and/or antagonist properties of anantibody secreted by an antibody secreting cell) on a readout cell. In afurther embodiment, the extracellular effect is an agonist or antagonisteffect on a transmembrane protein, which in a further embodiment is a Gprotein coupled receptor (GPCR), a receptor tyrosine kinase (RTK), anion channel or an ABC transporter. In a further embodiment, the receptoris a cytokine receptor. An extracellular effect on other metabotropicreceptors besides GPCRs and RTKs can be assessed, for example, anextracellular effect on a guanylyl cyclase receptor can be assessedincubating a cell population with a readout cell population expressingthe guanylyl cyclase receptor.

In embodiments where a readout cell is used, the readout cell can bealive or fixed. With respect to a fixed readout cell, the extracellulareffect in one embodiment, is an effect on an intracellular protein ofthe fixed readout cell. Extracellular effects can also be measured onextracellular proteins of an alive or fixed readout cell, or a secretedprotein of an alive readout cell.

In another embodiment, a readout cell expresses one of the followingtypes of cell receptors, and the extracellular effect assay measuresbinding, agonism or antagonism of the cell receptor: receptorserine/threonine kinase, histidine-kinase associated receptor.

In embodiments where a particular receptor (e.g., receptorserine/threonine kinase, histidine-kinase associated receptor or GPCR)is an orphan receptor, that is, the ligand for activating the particularreceptor is unknown, the methods provided herein allow for the discoveryof a ligand for the particular orphan receptor by performing anextracellular assay on readout cells expressing the orphan receptor, andidentifying a cell population or subpopulations comprising an effectorcell having a variation of an extracellular effect on the readout cellexpressing the orphan receptor.

In one embodiment, the cell surface protein is a transmembrane ionchannel. In a further embodiment, the ion channel is a ligand gated ionchannel and the extracellular effect measured in the microfluidic assayis modulation of the ion channel gating, for example, opening of the ionchannel by agonist binding or closing/blocking of the ion channel byantagonist binding. The antagonist or agonist can be for example, abiomolecule (e.g., antibody) secreted by one or more effector cells inthe heterogeneous population of cells comprising one or more effectorcells. Extracellular assays described herein can be used to measure theextracellular effect of an effector cell on a cell expressing a ligandgated ion channel in the Cys-loop superfamily, an ionotropic glutamatereceptor and/or an ATP gated ion channel. Specific examples of anioniccys-loop ion gated channels include the GABAA receptor and the glycinereceptor (GlyR). Specific examples of cationic cys-loop ion gatedchannels include the serotonin (5-HT) receptor, nicotinic acetylcholine(nAChR) and the zinc-activated ion channel. One or more of theaforementioned channels can be expressed by a readout cell to determinewhether an effector cell has an extracellular effect on the respectivecell by agonizing or antagonizing the ion channel. Ion flux measurementstypically occur in short periods of time (i.e., seconds) and requireprecise fluidic control for their implementation. Examples ofcommercially available ion channel assays include Fluo-4-Direct CalciumAssay Kit (Life Technologies), FLIPR Membrane Potential Assay Kit(Molecular Devices). Ion-channel expressing cell lines are alsocommercially available (e.g. PrecisION™ cell lines, EMD Millipore).

In one embodiment, the cell surface protein is an ATP-binding cassette(ABC) transporter, and the extracellular effect measured is thetransport of a substrate across a membrane. The readout particles can bemembrane vesicles derived from cells expressing the protein (e.g.,GenoMembrane ABC Transporter Vesicles (Life Technologies)), which can beimmobilized on beads. For instance, the ABC transporter could be apermeability glycoprotein (multidrug resistant protein) and the effectcan be measured by the fluorescence intensity of calcein in readoutcells. The Vybrant™ Multidrug Resistance Assay Kit (Molecular Probes) iscommercially available to implement such an assay.

An extracellular effect can also be assessed on a readout cellexpressing an ionotropic glutamate receptor such as the AMPA receptor(class GluA), kainite receptor (class GluK) or NMDA receptor (classGluN). Similarly, an extracellular effect can also be assessed on areadout cell expressing an ATP gated channel or a phosphatidylinositol4-5-bisphosphate (PIP2)-gated channel.

As provided throughout, the present invention provides a method ofidentifying a cell population displaying a variation in an extracellulareffect. In one embodiment, the method comprises, retaining a pluralityof individual cell populations in separate microfluidic chambers,wherein at least one of the individual cell populations comprises one ormore effector cells and the contents of the separate microfluidicchambers further comprise a readout particle population comprising oneor more readout particles, incubating the individual cell populationsand the readout particle population within the microfluidic chambers,assaying the individual cell populations for the presence of theextracellular effect, wherein the readout particle population orsubpopulation thereof provides a readout of the extracellular effect. Inone embodiment, the extracellular effect is an effect on a receptortyrosine kinase (RTK), for example, binding to the RTK, antagonism ofthe RTK, or agonism of the RTK. RTKs are high affinity cell surfacereceptors for many polypeptide growth factors, cytokines and hormones.To date, there have been approximately sixty receptor kinase proteinsidentified in the human genome (Robinson et al. (2000). Oncogene 19, pp.5548-5557, incorporated by reference in its entirety for all purposes).RTKs have been shown to regulate cellular processes and to have a rolein development and progression of many types of cancer (Zwick et al.(2001). Endocr. Relat. Cancer 8, pp. 161-173, incorporated by referencein its entirety for all purposes).

Where the extracellular effect is an effect on an RTK, the presentinvention is not limited to a specific RTK class or member.Approximately twenty different RTK classes have been identified, andextracellular effects on members of any one of these classes can bescreened for with the methods and devices provided herein. Table 2provides different RTK classes and representative members of each class,each amenable for use herein when expressed on a readout particle, e.g.,readout cell or vesicle. In one embodiment, a method is provided hereinfor screening a plurality of cell populations in a parallel manner inorder to identify one or more populations comprising an effector cellhaving an extracellular effect on an RTK of one of the subclassesprovided in Table 2. In one embodiment, the method further comprisesisolating the one or more cell populations comprising the ASC having theextracellular effect to provide an isolated cell population and furthersubjecting the isolated subpopulation to one or more additionalextracellular effect assay, at limiting dilution, to identify the ASChaving the extracellular effect. The additional extracellular effectassay can be carried out via a microfluidic method provided herein, or abenchtop assay. Alternatively, once a cell population is identified thathas a cell exhibiting an extracellular effect on the RTK, the cellpopulation is recovered, lysed and the nucleic acid amplified. In afurther embodiment, the nucleic acid is one or more antibody genes.

In one embodiment, the present invention relates to the identificationof a cell population comprising an effector cell that antagonizes oragonizes an RTK (i.e., the extracellular effect), for example, via asecretion product, e.g., a monoclonal antibody. The effector cell ispresent a lone effector cell, or is present in a cell populationcomprising one or more effector cells.

TABLE 2 RTK classes and representative members of each class.Representative Representative Cellular RTK class members LigandsProcess(es) RTK class I ErbB-1 (epidermal epidermal growthoverexpression (epidermal growth factor factor (EGF) implicated ingrowth factor receptor) transforming growth turmorigenesis receptorfactor α (TGF-α) (EGFR) heparin-binding family, also EGF-like growthknown as the factor (HB-EGF) ErbB family) amphiregulin (AREG)betacellulin epigen epiregulin ErbB-2 (human Monoclonal turmorigenesisepidermal growth antibody (e.g., breast, factor receptor 2 trastuzumabovarian, stomach, (HER2)/ cluster of (Herceptin) uterine)differentiation 340 (CD340) / proto- oncogene Neu) Neuregulin 1Proliferation and ErbB-3 (human Proliferation differentiation epidermalgrowth associated Oncogenesis factor receptor protein 2G4(overexpression) 2 (HER3)) (PA2G4) (EBP1 or ErbB3 binding protein 1)Phosphatidylinositol 3 kinase regulatory subunit alpha (PIK3R1)Regulator of G protein signaling 4 (RGP4) ErbB-4 (human heparin-bindingMutations in epidermal EGF-like growth the RTK have been growth factorfactor (HB- associated with receptor 2 (HER4)) EGF) betacellulin cancerepiregulin Neuregulin 1 Neuregulin 2 Neuregulin 3 Neuregulin 4 RTK classII Insulin receptor Insulin inducing glucose (Insulin insulin-likegrowth uptake receptor factor 1 (IGF-1) / family) somatomedin Cinsulin-like growth factor 2 (IGF-2) RTK class III PDGFRα PDGF A/B/C andD Fibrosis (Platelet PDGFRβ PDGF A/B/C and D cancer derived Mast/stemcell Stem cell factor Oncogenesis growth growth factor (SCF) / c-kitCell survival, factor receptor (SCFR) / ligand / steel factorproliferation, (PDGF) c-Kit / CD117 differentiation receptor Colonystimulating Colony stimulating Production, family) factor 1 factor 1differentiation and receptor / (CD 115) / function of macrophage colony-macrophages stimulating factor receptor (M-CSFR) Expressed on Cluster ofFlt3 ligand (FLT3L) surface of many differentiation hematopoieticantigen progenitor cells 135 (CD 135) / Mutated in acute Fms-liketyrosine myeloid leukemia kinase 3 (FLT-3) Cell survival Proliferationdifferentiation RTK class IV Fibroblast growth Fibroblast growth Woundhealing (FGF receptor factor receptor-1 factor 1-10 Embryonic family)(CD331) development Fibroblast angiogenesis growth factor receptor-2(CD332) Fibroblast growth factor receptor-3 (CD333) Fibroblast growthfactor receptor-4 (CD334) Fibroblast growth factor receptor-6 RTK classV VEGFR1 VEGF-A Mitogenesis (VEGF receptor VEGF-B Cell migration family)VEGFR2 VEGF-A Vasculogenesis (membrane VEGF-C angiogenesis bound VEGF-Dor soluble VEGF-E depending on VEGFR3 VEGF-C alternative VEGF-Dsplicing) RTK class VI Hepatocyte growth Hepatocyte growth Deregulatedin certain (Hepatocyte factor receptor factor malignancies, leads togrowth (GHFR) (encoded by angiogenesis factor MET or MNNG HOS Stem cellsand receptor transforming gene). progenitor cells express family)Mitogenesis, morphogenesis RTK class VII Tropomyosin- NeurotrophinsRegulate synaptic (Trk receptor receptor kinases Nerve growth factorstrength and family) (Trk) (TrkA) plasticity in TrkA Brain-derived themammalian TrkB neurotrophic factor nervous system TrkC (BDNF) (TrkB)Neurotrophin-3 (NT3) (TrkC) RTK class VIII EphA Ephrin-A Embryonicdevelopment (Ephrin (Eph) (1, 2, 3, 4, 5, 6, (Ephrin-A1-5) Axon guidancereceptor 7, 8, 9, 10) Ephrin-B (1-4 Formation of tissue family) EphB (1,2, 3, and ephrin-B6) boundaries 4, 5, 6) Retinopic mapping Cellmigration Cell segmentation Angiogenesis Cancer RTK class IXTyrosine-protein Tensin-like C1 epithelial-to- (AXL receptor kinasereceptor domain containing mesenchymal family) UFO (AXL) phosphatasetransition-induced (TENC1) regulator of breast cancer metastasisRegulation of cell migration RTK class X Leukocyte receptor Insulinreceptor Apoptosis (Leukocyte tyrosine kinase substrate 1 (IRS-1) Cellgrowth and receptor (LTK) Src homology 2 differentiation tyrosine kinasedomain containing (LTK) family) protein (Shc) Phosphatidylinositol3-kinase regulatory subunit alpha (PIK3R1) RTK class XI Tyrosine kinasewith Angiopoietin 1 Promotion of (TIE receptor immunoglobulin-like (Tie2agonist) angiogenesis TIE1 has family) and EGF-like Angiopoietin 2 aproinflammatory domains (TIE) 1 (Tie2 antagonist) effect and may play aTIE 2 Angiopoietin 3 role in atherosclerosis (Tie2 antagonist) (Chan etal. (2008). Angiopoietin 4 Biochem. Biophys. (Tie2 agonist) Res. Commun.371, pp. 475-479. RTK class XII ROR-1 (neurotrophic Wnt ligands ROR-1modulates (Receptor tyrosine kinase, (ROR-2) neurite growth typrosinekinase- receptor-related 1 in the central like orphan (NTRKR1) nervoussystem. receptors ROR-2 (ROR) family) RTK class XIII DDR-1 (CD167a)Various types of DDR-1 is (discoidin DDR-2 collagen overexpressed inbreast, domain ovarian, esophageal receptor and pediatric (DDR) family)brain tumors RTK class XIV Rearranged during Glial cell line- Loss offunction (RET receptor transfection derived associated with family)(RET) proto- nuerotrophic Hirschsprung's disease oncogene 3 differentfactor (GDNF) Gain of function isoforms (51, 43, 9) family ligandsmutations associated with various types of cancer (e.g., medullarythyroid carcinoma, multiple endocrine neoplasias type 2A and 2D) RTKclass XV Tyrosine-protein No ligand has Development (KLG receptorkinase-like 7 been identified Oncogenesis (colon family) (PTK7) / CCK-4cancer, melanoma, breast cancer, acute myeloid leukemia) Wnt pathwayregulation Angiogenesis RTK class XVI RYK receptor Wnt ligandsStimulating (Related to (different Wnt signaling receptor tyrosineisoforms due to pathways such kinase(RYK) alternative splicing) asregulation of receptor family) axon pathfinding RTK class XVIIMuscle-Specific Agrin (nerve- Formation of the (Muscle-Specific kinase(MuSK) derived neuromuscular kinase (MuSK) receptor proteoglycan)junction receptor family)

In one embodiment, the RTK is a platelet derived growth factor receptor(PDGFR), e.g., PDGFRα. PDGFs are a family of soluble growth factors (A,B, C, and D) that combine to form a variety of homo- and hetero-dimers.These dimers are recognized by two closely related receptors, PDGFRα andPDGFRβ, with different specificities. In particular, PDGF-α bindsselectively to PDGFRα and has been shown to drive pathologicalmesenchymal responses in fibrotic diseases, including pulmonaryfibrosis, liver cirrhosis, scleroderma, glomerulosclerosis, and cardiacfibrosis (see Andrae et al. (2008). Genes Dev. 22, pp. 1276-1312,incorporated by reference herein in its entirety). It has also beendemonstrated that constitutive activation of PDGFRα in mice leads toprogressive fibrosis in multiple organs (Olson et al. (2009). Dev. Cell16, pp. 303-313, incorporated by reference herein in its entirety). Thustherapies that inhibit PDGFRα have high potential for the treatment offibrosis, a condition that complicates up to 40% of diseases, andrepresents a huge unmet medical problem in the aging population.Although antibodies (Imatinib and Nilotinib) have been explored asinhibitors of PDGFRα, each has significant off-target effects on othercentral RTKs, including c-kit and Flt-3, resulting in numerous sideeffects. Thus, while Imatinib and Nilotinib can effectively inhibitPDGFRα and PDGFRβ, their side effects make them unacceptable in thetreatment of fibrotic diseases, highlighting the potential for highlyspecific antibody inhibitors. The present invention overcomes thisproblem by providing in one embodiment, antibodies with greater PDGFRαspecificity, as compared to Imatinib and Nilotinib.

PDGFRα has been previously established as a target for the treatment offibrosis. Two anti-human PDGFRα mAb antagonists entering early clinicaltrials for the treatment of cancer are in development (see, e.g., Shahet al (2010). Cancer 116, pp. 1018-1026, incorporated by referenceherein in its entirety). The methods provided herein facilitate theidentification of an effector cell secretion product that binds to thePDGFRα. In a further embodiment, the secretion product blocks theactivity of both human and murine PDGFRα in both cancer and fibrosismodels.

One embodiment of the effector cell assay to determine whether aneffector cell secretion product binds to PDGFRα is based on the use ofsuspension cell lines (e.g., 32D and Ba/F3) that are strictly dependenton the cytokine IL-3 for survival and growth, but can be cured of this“IL-3 addiction” through the expression and activation of nearly anytyrosine kinase. This approach was first used by Dailey and Baltimore toevaluate the BCR-ABL fusion oncogene and has been used extensively forhigh-throughput screening of small molecule tyrosine kinase inhibitors(see, e.g., Warmuth et al. (2007). Curr. Opin. Oncology 19, pp. 55-60;Daley and Baltimore (1988). Proc. Natl. Acad. Sci. U.S.A. 85, pp.9312-9316, each incorporated by reference in their entireties for allpurposes). To monitor signaling, PDGFRα and PDGFRβ (both human and mouseforms) are expressed in 32D cells (readout cells), a murinehematopoietic cell line that does not naturally express either receptor.This allows for separation of each pathway, something that is otherwisedifficult since both receptors are often co-expressed. Expression ofhuman PDGFRα/β in 32D cells has been previously confirmed to give afunctional PDGF-induced mitogenic response (Matsui et al. (1989). Proc.Natl. Acad. Sci. U.S.A. 86, pp. 8314-8318, incorporated by reference inits entirety). In the absence of IL-3, 32D cells do not divide at all,but PDGF stimulation of the cells expressing the RTK relieves therequirement for IL-3 and gives a rapid mitogenic response that isdetectable by microscopy. The detectable response, in one embodiment, iscell proliferation, a morphological change, increasedmotility/chemotaxis, or cell death/apoptosis in the presence of anantagonist. An optical multiplexing method, in one embodiment, is usedto simultaneously measure the inhibition/activation of both PDGFRα andPDGFRβ responses in one of the devices provided herein. In anotherembodiment, inhibition/activation of both PDGFRα and PDGFRβ responses inone of the devices provided herein is measured by two extracellularassays, carried out serially in the same microfluidic chamber.

Full length cDNA for human/mouse PDGFRα and PDGFRβ (Sino Biological), inone embodiment, is expressed in 32D cells (ATCC; CRL-11346) usingmodified pCMV expression vectors that also include an IRES sequence witheither GFP or RFP, thereby making two types of “readout cells,” eachdistinguishable by fluorescent imaging. The readout cells arecharacterized to optimize medium and feeding conditions, determine thedose response to PDGF ligand, and to characterize the morphology andkinetics of response. The use of suspension cells (such as 32D or Ba/F3)provides the advantage that single cells are easily identified by imageanalysis, and are also physically smaller (in projected area) thanadherent cells so that a single chamber can accommodate ≥100 readoutcells before reaching confluence. In another embodiment, instead of 32Dcells, Ba/F3 cells, another IL-3 dependent mouse cell line with similarproperties to 32D are used as readout cells. Both 32D and Ba/F3 cellsare derived from bone marrow, grow well in medium optimized for ASCs,and also secrete IL-6 which is a critical growth factor for themaintenance of ASCs (see, e.g., Cassese et al. (2003). J. Immunol. 171,pp. 1684-1690, incorporated by reference in its entirety herein).

Preclinical models for evaluating the role of PDGFR□□ in fibrosis havebeen developed. Specifically, two models of cardiac fibrosis areprovided herein. The first is based on ischemic damage(isoproterenol-induced cardiac damage; ICD) and the second is based oncoronary artery ligation-induced myocardial infarction (MI). Upondamage, the fibrotic response is initiated by the rapid expansion ofPDGFRα+/Sca1+ positive progenitors, accounting for over 50% of the cellsproliferating in response to damage followed by differentiation of theseprogeny into matrix producing PDGFRα low/Sca1 low myofibroblasts. Geneexpression by RT-qPCR demonstrates expression of multiple markersrelated to fibrotic matrix deposition, including α-smooth muscle actin(aSMA) and collagen type I (Coll), which are detectable in (Sca1+)progenitors, but are substantially up-regulated in the differentiatedpopulation. In one embodiment, a cell population is identified thatcomprises an effector cell that secretes a monoclonal antibody thatattenuates progenitor expansion leading to reduced fibrosis. Thisextracellular effect assay is carried out by monitoring two independentmarkers: early proliferation of Sca1+/PDGFRα+ progenitors cells andColl-driven GFP. Specifically, following MI, fibrotic responses arecharacterized by GFP expression first in PDGFRα+/Sca1+ progenitors, andlater, with increased intensity, in the emerging myofibroblastpopulation.

The present invention provides methods and devices for screening aplurality of cell populations in a parallel manner to identify one ormore of the cell populations having an extracellular effect, or avariation in an extracellular effect as compared to another populationin the plurality. The identified cell population comprises one or moreeffector cells that are responsible for the extracellular effect. Theextracellular effect, for example, is an extracellular effect on a GPCR,e.g., GPCR binding, agonism or antagonism. As described herein, theextracellular effect need not be attributable to every cell in thepopulation, or even multiple cells. Rather, the methods provided hereinallow for the detection of an extracellular effect of a single effectorcell, when the effector cell is present in a heterogeneous populationcomprising tens to hundreds of cells (e.g., from about 10 to about 500cells, or from about 10 to about 100 cells), or comprising from about 2to about 100 cells, e.g., from about 2 to about 10 cells.

GPCRs are a superfamily of seven transmembrane receptors that includesover 800 members in the human genome. Each GPCR has its amino terminuslocated on the extracellular face of the cell and the C-terminal tailfacing the cytosol. On the inside of the cell GPCRs bind toheterotrimeric G-proteins. Upon agonist binding, the GPCR undergoes aconformational change that leads to activation of the associatedG-protein. Approximately half of these are olfactory receptors with therest responding to a gamut of different ligands that range from calciumand metabolites to cytokines and neurotransmitters. The presentinvention, in one embodiment, provides a method for selecting one ormore ASCs that have an extracellular effect on a GPCR. The GPCR is notlimited herein. Rather, screening methods for any GPCR are amenable foruse with the present invention.

The type of G-protein that naturally associates with the specific GPCRdictates the cell signaling cascade that is transduced. For Gq coupledreceptors the signal that results from receptor activation is anincrease in intracellular calcium levels. For Gs coupled receptors, anincrease in intracellular cAMP is observed. For Gi coupled receptors,which make up 50% of all GPCRs, activation results in an inhibition ofcAMP production. For embodiments where the effector cell property isactivation of a Gi coupled GPCR, it is sometimes necessary to stimulatethe readout cell(s) with a nonspecific activator of adenylyl cyclase. Inone embodiment, the adenyl cyclase activator is forskolin. Thus,activation of the Gi coupled receptor by one or more effector cells willprevent forskolin induced increase in cAMP. Forskolin, accordingly, canbe used as an accessory particle in one or more GPCR extracellulareffect assays provided herein.

The present invention, in one embodiment, provides means for determiningwhether an effector cell (e.g., an ASC) within a cell population has anextracellular effect on a GPCR. The GPCR is present on one or morereadout particles in a microfluidic chamber and the extracellular effectin one embodiment, is binding to the GPCR, a demonstrated affinity orspecificity, inhibition or activation. The GPCR may be a stabilizedGPCR, such as one of the GPCRs made by the methods of HeptaresTherapeutics (stabilized receptor, StaR® technology). The effector cell(e.g., ASC), in one embodiment, is present as a single cell, or in ahomogeneous or heterogeneous cell population within a microfluidicchamber. In one embodiment, the methods and devices provided herein areused to identify one or more cell populations each comprising one ormore ASCs that secrete one or more antibodies that demonstrate anextracellular effect on one of the GPCRs set forth in Table 3A and/orTable 3B, or one of the GPCRs disclosed in International PCT PublicationWO 2004/040000, incorporated by reference in its entirety. For example,in one embodiment, the GPCR belongs to one of the following classes:class A, class B, class C, adhesion, frizzled.

In another embodiment, the extracellular effect is an effect on anendothelial differentiation, G-protein-coupled (EDG) receptor. The EDGreceptor family includes II GPCRs (S1P1-5 and LPA1-6) that areresponsible for lipid signalling and bind lysophosphandic acid (LPA) andsphingosine I-phosphate (SIP). Signalling through LPA and SIP regulatesnumerous functions in health and disease, including cell proliferation,immune cell activation, migration, invasion, inflammation, andangiogenesis. There has been little success in generating potent andspecific small molecule inhibitors to this family, making mAbs a veryattractive, alternative. In one embodiment, the EDG receptor is S1P3(EDG3), S1PR1 (EDG1), the latter of which has been shown to activateNF-κB and STAT3 in several types of cancers including breast, lymphoma,ovarian, and melanoma, and plays a key role in immune cell traffickingand cancer metastasis (Milstien and Spiegel (2006). Cancer Cell 9, pp.148-15, incorporated by reference in its entirety herein). A monoclonalantibody that neutralizes the SIP ligand (Sonepcizumab) has recentlyentered phase II trials for treatment of advanced solid tumours(NCT00661414). In one embodiment, the methods and devices providedherein are used to identify and isolate an ASC that secretes an antibodywith greater affinity than Sonepcizumab, or an antibody that inhibitsSIP to a greater extent than Sonepcizumab. In another embodiment, theextracellular effect is an effect on the LPA2 (EDG4) receptor. LPA2 isoverexpressed in thyroid, colon, stomach and breast carcinomas, as wellas many ovarian tumours, for which LPA2 is the primary contributor tothe sensitivity and deleterious effects of LPA.

In one embodiment, cell populations are assayed for whether they exhibitan extracellular effect on a chemokine receptor, present on a readoutparticle. In a further embodiment, the chemokine receptor is C-X-Cchemokine receptor type 4 (CXCR-4), also known as fusin or CD184. CXCR4binds SDF1α (CXCL12), a strong chemotactic for immune cell recruitmentalso known as C-X-C motif chemokine 12 (CXCL12). DNA immunizations wereused to generate 92 hybridomas against this target, 75 of whichexhibited different chain usage and epitope recognition (Genetic Eng andBiotech news, August 2013), indicating that hybridoma selections captureonly a small portion of available antibody diversity. Signalling throughthe CXCR4/CXCL12 axis has been shown to play a central role in tumourcell growth, angiogenesis, cell survival and to be implicated inmediating the growth of secondary metastases in CXCL12-producing organslike liver and bone marrow (Teicher and Fricker (2010). Clin. CancerRes. 16, pp. 2927-2931, incorporated by reference herein in itsentirety).

In another embodiment, cell populations are screened for their abilityto exert an effect on the chemokine receptor CXCR7, which was recentlyfound to bind SDF1α. Unlike CXCR4 which signals through canonical Gprotein coupling, CXCR7 signals uniquely through the 3-arrestin pathway.

In another embodiment, the GPCR is protease activated receptors (PAR1,PAR3, and PAR4), which is a class of GPCRs activated bythrombin-mediated cleavage of the exposed N-terminus and are involved infibrosis. In yet another embodiment, the GPCR is one of the GPCRs inTable 3A or Table 3B, below.

In embodiments where an extracellular effect is an effect on a GPCR, theinvention is not limited to the particular GPCR. For example, cell linesthat express particular GPCRs, engineered to provide a readout ofbinding, activation or inhibition, are commercially available forexample, from Life Technologies (GeneBLAzer® and Tango™ cell lines),DiscoveRx, Cisbio, Perkin Elmer, etc., and are amenable for use asreadout cells herein.

In one embodiment, a GPCR from one of the following receptor families isexpressed on one or more readout cells herein, and an extracellulareffect is measured with respect to one or more of the following GPCRs:acetylcholine receptor, adenosine receptor, adreno receptor, angiotensinreceptor, bradykinin receptor, calcitonin receptor, calcium sensingreceptor, cannabinoid receptor, chemokine receptor, cholecystokininreceptor, complement component (C5AR1), corticotrophin releasing factorreceptor, dopamine receptor, endothelial differentiation gene receptor,endothelin receptor, formyl peptide-like receptor, galanin receptor,gastrin releasing peptide receptor, receptor ghrelin receptor, gastricinhibitory polypeptide receptor, glucagon receptor, gonadotropinreleasing hormone receptor, histamine receptor, kisspeptin (KiSS1)receptor, leukotriene receptor, melanin-concentrating hormone receptor,melanocortin receptor, melatonin receptor, motilin receptor,neuropeptide receptor, nicotinic acid, opioid receptor, orexin receptor,orphan receptor, platelet activating factor receptor, prokineticinreceptor, prolactin releasing peptide, prostanoid receptor, proteaseactivated receptor, P2Y (purinergic) receptor, relaxin receptor,secretin receptor, serotonin receptor, somatostatin receptor, tachykininreceptor, vasopressin receptor, oxytocin receptor, vasoactive intestinalpeptide (VIP) receptor or the pituitary adenylate cyclase activatingpolypeptide (PACAP) receptor.

TABLE 3A GPCRs amenable for expression in Readout Cells or as Part of aStabilized Readout Particle. Human gene Family symbol Human Gene NameCalcitonin receptors CALCR calcitonin receptor Calcitonin receptorsCALCRL calcitonin receptor-like Corticotropin- CRHR1 corticotropinreleasing releasing hormone factor receptors receptor 1 Corticotropin-CRHR2 corticotropin releasing releasing hormone factor receptorsreceptor 2 Glucagon receptor GHRHR growth hormone family releasinghormone receptor Glucagon receptor GIPR gastric inhibitory polypeptidefamily receptor Glucagon receptor GLP1R glucagon-like family peptide 1receptor Glucagon receptor GLP2R glucagon-like family peptide 2 receptorGlucagon receptor GCGR glucagon receptor family Glucagon receptor SCTRsecretin receptor family Parathyroid PTH1R parathyroid hormone hormonereceptors 1 receptor Parathyroid PTH2R parathyroid hormone hormonereceptors 2 receptor VIP and PACAP ADCYAP1R1 adenylate cyclaseactivating receptors polypeptide 1 (pituitary) receptor type I VIP andPACAP VIPR1 vasoactive intestinal receptors peptide receptor 1 VIP andPACAP VIPR2 vasoactive intestinal receptors peptide receptor 2 AdenosineADORA1 Adenosine A1 receptor Adenosine ADORA2A Adenosine A2 receptorAdenosine ADRB3 Adenosine 3 receptor Chemokine CXCR1 C-X-C chemokinereceptor 1 Chemokine CXCR2 C-X-C chemokine receptor 2 Chemokine CXCR3C-X-C chemokine receptor 3 Chemokine CXCR4 C-X-C chemokine receptor 4Chemokine CXCR5 C-X-C chemokine receptor 5 Chemokine CXCR6 C-X-Cchemokine receptor 6 Chemokine CXCR7 C-X-C chemokine receptor 7Chemokine CCR1 C-C chemokine receptor type 1 Chemokine CCR2 C-Cchemokine receptor type 2 Chemokine CCR3 C-C chemokine receptor type 3Chemokine CCR4 C-C chemokine receptor type 4 Chemokine CCR5 C-Cchemokine receptor type 5 Chemokine CCR6 C-C chemokine receptor type 6Chemokine CCR7 C-C chemokine receptor type 7 Chemokine CMKLR1 Chemokinereceptor-like 1 Complement C5AR1 Complement component AR1 componentreceptor Lysophospholipid LPAR1 lysophosphatidic acid (LPL) receptorreceptor 1 Lysophospholipid LPAR2 lysophosphatidic acid (LPL) receptorreceptor 2 Lysophospholipid LPAR3 lysophosphatidic acid (LPL) receptorreceptor 3 Lysophospholipid LPAR4 lysophosphatidic acid (LPL) receptorreceptor 4 Lysophospholipid LPAR5 lysophosphatidic acid (LPL) receptorreceptor 5 Lysophospholipid LPAR6 lysophosphatidic acid (LPL) receptorreceptor 6 Lysophospholipid SIPR1 sphingosine-1- (LPL) receptorphosphate receptor 1 Lysophospholipid SIPR2 sphingosine-1- (LPL)receptor phosphate receptor 2 Lysophospholipid SIPR3 sphingosine-1-(LPL) receptor phosphate receptor 3 Lysophospholipid SIPR4sphingosine-1- (LPL) receptor phosphate receptor 4 LysophospholipidSIPR5 sphingosine-1- (LPL) receptor phosphate receptor 5

TABLE 3B GPCRs amenable for expression in Readout Cells or as Part of aStabilized Readout Particle. GPCR (Gene symbol) Ligand(s)5-hydroxytryptamine 5-hydroxytryptamine 1A receptor (HTR1A)5-hydroxytryptamine 5-hydroxytryptamine 1B receptor (HTR1B)5-hydroxytryptamine 5-hydroxytryptamine 1D receptor (HTR1D)5-hydroxytryptamine 5-hydroxytryptamine 1e receptor (HTR1E)5-hydroxytryptamine 1F 5-hydroxytryptamine receptor (HTR1F)5-hydroxytryptamine 2A 5-hydroxytryptamine receptor (HTR2A)5-hydroxytryptamine 2B 5-hydroxytryptamine receptor (HTR2B)5-hydroxytryptamine 2C 5-hydroxytryptamine receptor (HTR2C)5-hydroxytryptamine 4 5-hydroxytryptamine receptor (HTR4)5-hydroxytryptamine 5a 5-hydroxytryptamine receptor (HTR5A)5-hydroxytryptamine 5b 5-hydroxytryptamine receptor (HTR5BP)5-hydroxytryptamine 5-hydroxytryptamine 6 receptor (HTR6)5-hydroxytryptamine 7 5-hydroxytryptamine receptor (HTR7) AcetylcholineM1 acetylcholine receptor (CHRA11) Acetylcholine M2 acetylcholinereceptor (CHR1142) Acetylcholine M3 acetylcholine receptor (CHRI143)Acetylcholine M4 acetylcholine receptor (CHRI144) Acetylcholine M5acetylcholine receptor (CHRI145) Adenosine Al receptor adenosine(ADORA1) Adenosine A2A receptor (ADORA2A) adenosine Adenosine A2Breceptor (ADORA2B) adenosine Adenosine A3 receptor adenosine (ADORA3)α_(1A)-adrenoceptor Adrenaline, noradrenaline (ADRA1A) agonistscirazoline, desvenlafaxine, etilefrine, metaraminol, methoxamine,midodrine, naphazoline, oxymetrazoline, phenylephrine, synephrine,tetrahydrozoline, xylometazoline antagonists alfuzosin, arotinolol,carvedilol, doxazosin, indoramin, labetalol, moxislyte,phenoxybenzamine, phentolamine, prazosin, quetiapine, risperidone,silodosin, tamsulosin, terazosin, tolazoline, trimazosinα_(1B)-adrenoceptor Adrenaline, noradrenaline (ADRA1B)α_(1D)-adrenoceptor Adrenaline (ADRA1D) α_(2A)-adrenoceptor Adrenaline(ADRA2A) α_(2C)-adrenoceptor Adrenaline, noradrenaline (ADRA2B) agonistssalbutamol, bitolterol mesylate, isoproteronol, levosalbutamol,metaproterenol, formoterol, salmeterol, terbutaline, clenbuterol,ritodrine antagonists butoxamine, Beta blockers a_(2C)-adrenoceptorAdrenaline, noradrenaline (ADRA2C) β₁-adrenoceptor Adrenaline,noradrenaline (ADRB1) nitrosamine 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) agonists denopamine, dobutamine,xamoterol antagonists acebutol, atenolol, betaxolol, bisoprolol,esmolol, metoprolol, nebivolol, vortioxetine β₂-adrenoceptor Adrenaline(ADRB2) β₃-adrenoceptor (ADRB3) Adrenaline Angiotensin recptor 1Angiotensisn I, angiotensin II, (AT₁) (AGTR1) angiotensin IIIAngiotensin recptor 2 Angiotensisn II, angiotensin III (AT₂) (AGTR2)Angiotensin recptor 4 Angiotensisn IV (angiotensin II metabolite) Apelinreceptor (APLNR) Apelin-13, apelin-17, apelin-36 Bile acid receptorChenodeoxycholic acid, (GPBAR1) cholic acid, deoxycholic acid,lithocholic acid Bombesin receptor BB₁ (NMBR) Gastrin-releasing peptide,neuromedin B Bombesin receptor BB₂ Gastrin-releasing peptide, (GRPR)neuromedin B Bombesin receptor BB₃ (BRS3) Bradykinin recpetor B1bradykinin Bradykinin recpetor B2 bradykinin Calcium sensing Calciumreceptor (CaSR) Magnesium G-protein coupled receptor family C group 6member A (GPRC6A) Cannabinoid CB₁ receptor 2-arachidonoylglycerol (CNR1)anandamide Cannabinoid CB₂ receptor 2-arachidonoylglycerol (CNR2)anandamide C-X-C chemokine receptor Interleukin 8 type 4 (CXCR2)Growth-related oncogene- alpha (GRO-α) C-X-C chemokine receptor Stromalcell-derived type 4 (CXCR4) factor-1 (SDF1) Cholecystokinin A receptorCholecystokinin peptide (CCKAR or CCK1) hormones (CCK) Cholecystokinin Breceptor Cholecystokinin peptide (CCKAR or CCK2) hormones(CCK) GastrinCholecystokinin recptor CCK-33, CCK-4, CCK-8, CCK1 (CCKAR) gastrin-17Cholecystokinin recptor CCK-33, CCK-4, CCK-8, CCK2 (CCKBR) gastrin-17endothelin receptor A (ETA) Endothelin-1 endothelin receptor B1Endothelin-1 (ETB1) Endothelin-3 endothelin receptor B2 Endothelin-1(ETB2) Endothelin-3 endothelin receptor C (ETC) Endothelin-1 Frizzled 1(FZD1) Wnt-1, Wnt-2, Wnt-3a, Wnt-5a, Wnt-7b Frizzled 2 (FZD2) Wnt-5aFrizzled 3 (FZD3) Wnt protein ligand Frizzled 4 (FZD4) Wnt proteinligand Frizzled 5 (FZD5) Wnt protein ligand Frizzled 6 (FZD6) Wnt-3a,Wnt-4, Wnt-5a Frizzled 7 (FZD7) Wnt protein ligand Frizzled 8 (FZD8) Wntprotein ligands Frizzled 9 (FZD9) Wnt protein ligands Frizzled 10(FZD10) Wnt protein ligands GABA_(B1) Receptor Agonist GABA_(B2)Receptor GABA, Baclofen, gamma- (B1 and B2 assemble as hydroxybutyrate,phenibut, heterodimer) 3-aminopropylphosphinic acid, lesogaberan,SKF-97541, CGP-44532 Allosteric modulator CGP-7930, BHFF, Fendiline,BHF-177, BSPP, GS-39783 Antagonists 2-OH-saclofen, saclofen, phaclofen,SCH-50911, CGP-35348, CGP-52432, SGS-742, CGP-55845 Gastrin-releasingpeptide Gastrin releasing peptide receptor (GRPR), also referred to asBB₂ G protein-coupled estrogen Oestrogen receptor 30 (GPR30) Luteinizinghormone/ Luteinizing hormone choriogonadotropin receptor Chroinicgonadotropins (LHCGR), also referred to as Lutenizing hormone receptor(LHR) and lutropin/ choriogonadotroptin receptor (LCGR) Lysophosphatidicacid Lysophosphatidic acid receptor 1 (LPA1) Lysophosphatidic acidLysophosphatidic acid receptor 2 (LPA2) Lysophosphatidic acidLysophosphatidic acid receptor 3 (LPA3) Melanocortin 1 receptorMelanocortins (pituitary (MC1R), also referred peptide hormones)including o as melanocyte- adrenocorticotropic hormone stimulatinghormone (ACTH) and melanocyte- receptor (MSHR), melanin- stimulatinghormone (MSH) activating peptide receptor and melanotropin receptorNeuromedin B receptor Neuromedin B Prostaglandin E2 Prostaglandin E2receptor EP2 Prostaglandin E2 Prostaglandin E2 receptor EP4Protease-activated receptor 1 Thrombin Protease-activated receptor 2Trypsin Protease-activated receptor 3 Thrombin Protease-activatedreceptor 4 Thrombin Smoothened Sonic hedgehog Thyrotropin receptorThyrotropin (TSH receptor) Metabotropic glutamate L-glutamic acidreceptor 1(GRM1) Metabotropic glutamate L-glutamic acid receptor 2(GRM2) Metabotropic glutamate L-glutamic acid receptor 3 (GRM3)Metabotropic glutamate L-glutamic acid receptor 4 (GRM4) Metabotropicglutamate L-glutamic acid receptor 5 (GRM5) Metabotropic glutamateL-glutamic acid receptor 6 (GRM6) Metabotropic glutamate L-glutamic acidreceptor 7 (GRM7) Metabotropic glutamate L-glutamic acid receptor 8(GRM8) G protein-coupled receptor 56 (GPR56) G protein-coupled receptor64 (GPR64) G protein-coupled receptor 97 (GPR97) G protein-coupledreceptor 98 (GPR98) G protein-coupled receptor 110 (GPR110) Gprotein-coupled receptor 111 (GPR111) G protein-coupled receptor 112(GPR112) G protein-coupled receptor 113 (GPR113) G protein-coupledreceptor 114 (GPR114) G protein-coupled receptor 115 (GPR115) Gprotein-coupled receptor 116 (GPR116) G protein-coupled receptor 123(GPR123) G protein-coupled receptor 124 (GPR124) G protein-coupledreceptor 125 (GPR125) G protein-coupled receptor 126 (GPR126) Gprotein-coupled receptor 128 (GPR128) G protein-coupled receptor 133(GPR133) G protein-coupled receptor 144 (GPR144) latrophilin 1 (LPHN1)latrophilin 2 (LPHN2) latrophilin 3 (LPHN3)

In one embodiment, an effector cell is assayed for an extracellulareffect on a readout cell expressing a GPCR by one or more of the assaysprovided in Table 4, below. In another embodiment, a readout particlepopulation comprises a vesicle or a bead functionalized with a membraneextracts (available from Integral Molecular), or a stabilizedsolubilized GPCR (e.g., Heptares).

GPCRs can be phosphorylated and interact with proteins called arrestins.The three major ways to measure arrestin activation are: (i)microscopy—using a fluorescently labeled arrestin (e.g., GFP or YFP);(ii) using enzyme complementation; (iii) using the TANGO™ Reportersystem (β-lactamase) (Promega). In one embodiment, the TANGO™ Reportersystem is employed in a readout cell or plurality of readout cells. Thistechnology uses a GPCR linked to a transcription factor through acleavable linker. The arrestin is fused to a crippled protease. Once thearrestin binds to the GPCR, the high local concentration of the proteaseand the linker result in cleavage of the linker, releasing thetranscription factor into the nucleus to activate transcription. Theβ-lactamase assay can be run on live cells, does not require cell lysis,and can be imaged in as little as 6-hours of agonist incubation.

In one embodiment, a β-arrestin GPCR assay that can be universally usedfor the detection of antagonists and agonists of GPCR signaling is usedin the methods and devices provided herein to identify and an effectorcell that secretes a biomolecule that binds to a GPCR (Rossi et al.(1997). Proc. Natl. Acad. Sci. U.S.A. 94, pp. 8405-8410, incorporated byreference in its entirety for all purposes). This assay is based on aβ-galactosidase (β-Gal) enzyme-complementation technology, nowcommercialized by DiscoveRx. The GPCR target is fused in frame with asmall N-terminal fragment of the β-Gal enzyme. Upon GPCR activation, asecond fusion protein, containing β-arrestin linked to the N-terminalsequences of β-Gal, binds to the GPCR, resulting in the formation of afunctional β-Gal enzyme. The β-Gal enzyme then rapidly convertsnon-fluorescent substrate Di-β-D-Galactopyranoside (FDG) to fluorescein,providing large amplification and excellent sensitivity. In thisembodiment, readout cells (with GPCRs) are preloaded, off chip, withcell-permeable pro-substrate (acetylated FDG) which is converted tocell-impermeable FDG by esterase cleavage of acetate groups. Althoughfluorescein is actively transported out of live cells, by implementingthis assay within a microfluidic chamber the fluorescent product isconcentrated, providing greatly enhanced sensitivity over plate-basedassays. DiscoveRx has validated this assay strategy, used in microwellformat, across a large panel of GPCRs.

In one embodiment, activation of a GPCR by an effector cell isdetermined in a microfluidic format by detecting the increase incytosolic calcium in one or more readout cells. In a further embodiment,the increase in cytosolic calcium is detected with one or more calciumsensitive dyes. Calcium sensitive dyes have a low level of fluorescencein the absence of calcium and undergo an increase in fluorescentproperties once bound by calcium. The fluorescent signal peaks at aboutone minute and is detectable over a 5 to 10 minute window. Thus, todetect activity using fluorescent calcium the detection and addition ofthe agonist are closely coupled. In order to achieve this coupling, theeffector cell is exposed simultaneously to the population of readoutcells and the one or more calcium sensitive dyes. In one embodiment, theone or more calcium sensitive dyes are one provided in a FLIPR™ calciumassay (Molecular Devices).

The recombinant expressed jellyfish photoprotein, aequorin, in oneembodiment, is used in a functional GPCR screen, i.e., an extracellulareffect assay where the extracellular effect is the modulation of a GPCR.Aequorin is a calcium-sensitive reporter protein that generates aluminescent signal when a coelenterazine derivative is added. Engineeredcell lines with GPCRs expressed with a mitochondrially targeted versionof apoaequorin are available commercially (Euroscreen). In oneembodiment, the one or more of the cell lines available from Euroscreenis used as a population of readout cells in a method of assessing anextracellular effect of an effector cell, or a variation in anextracellular effect.

In one embodiment, an extracellular effect on a GPCR is measured byusing one of the ACTOne cell lines (Codex Biosolutions), expressing aGPCR and a cyclic nucleotide-gated (CNG) channel, as a population ofreadout cells. In this embodiment, the extracellular effect assay workswith cell lines that contain an exogenous Cyclic Nucleotide-Gated (CNG)channel. The channel is activated by elevated intracellular levels ofcAMP, which results in ion flux (often detectable by calcium-responsivedyes) and cell membrane depolarization which can be detected with afluorescent membrane potential (MP) dye. The ACTOne cAMP assay allowsboth end-point and kinetic measurement of intracellular cAMP changeswith a fluorescence microplate reader.

A reporter gene assay, in one embodiment, is used to determine whetheran effector cell modulates a particular GPCR. In this embodiment, themodulation of the GPCR is the extracellular effect being assessed. Areporter gene assay, in one embodiment, is based on a GPCR secondmessenger such as calcium (AP1 or NFAT response elements) or cAMP (CREresponse element) to activate or inhibit a responsive element placedupstream of a minimal promoter, which in turn regulates the expressionof the reporter protein chosen by the user. Expression of the reporter,in one embodiment, is coupled to a response element of a transcriptionfactor activated by signaling through a GPCR. For example, reporter geneexpression can be coupled to a responsive element for one of thefollowing transcription factors: ATF2/ATF3/AFT4, CREB, ELK1/SRF,FOS/JUN, MEF2, GLI, FOXO, STAT3, NFAT, NFκB. In a further embodiment,the transcription factor is NFAT. Reporter gene assays are availablecommercially, for example from SA Biosciences

Reporter proteins are known in the art and include, for example,β-galactosidase, luciferase (see, e.g., Paguio et al. (2006). “UsingLuciferase Reporter Assays to Screen for GPCR Modulators,” Cell NotesIssue 16, pp. 22-25; Dual-Glo™ Luciferase Assay System Technical Manual#TM058; pGL4 Luciferase Reporter Vectors Technical Manual #TM259, eachincorporated by reference in their entireties for all purposes), GFP,YFP, CFP, β-lactamase. Reporter gene assays for measuring GPCR signalingare available commercially and can be used in the methods and devicesdescribed herein. For example, the GeneBLAzer® assay from LifeTechnologies is amenable for use with the present invention.

In one embodiment, overexpression of a G protein in a reporter cell iscarried out to force a cAMP coupled GPCR to signal through calcium. Thisis referred to as force coupling.

In one embodiment, a Gq coupled cell line is used as a readout cell linein the methods described herein. In one embodiment, the Gq coupled cellline reports GPCR signaling through β-lactamase. For example, one of thecell-based GPCR reporter cell lines (GeneBLAzer®, Life Technologies).The reporter cell line can be division arrested or include normaldividing cells.

cAMP responsive element-binding protein (CREB) is a transcription factoras mentioned above, and is used in one embodiment for a Gs and/or Gicoupled GPCR. In a further embodiment, forskolin is utilized as anaccessory particle. The CRE reporter is available in plasmid orlentiviral form to drive GFP expression from SA Biosciences, and isamenable for use with the methods and devices described herein. Forexample, the assay system available from SA Biosciences in oneembodiment is employed herein to produce a readout cell(world-wide-website: sabiosciences.com/reporterassayproduct/HTML/CCS-002G.html). Life Technologies also hasCRE-responsive cell lines that express specific GPCRs, and these can beused in the methods described herein as well as readout cells.

In one embodiment, one or more effector cells present in a cellpopulation are assayed for the ability to activate or antagonize a GPCRpresent on one or more readout cells by detecting the increase ordecrease in cAMP levels inside the one or more readout cells. ELISAbased assays, homogeneous time-resolved fluorescence (HTRF) (see Degorceet al. (2009). Current Chemical Genomics 3, pp. 22-32, the disclosure ofwhich is incorporated by reference in its entirety), and enzymecomplementation can all be used with the microfluidic devices and assaysprovided herein to determine cAMP levels in readout cells. Each of thesecAMP detection methods requires cell lysis to liberate the cAMP fordetection, as it is the cyclic AMP that is actually measured.

Assays for measuring cAMP in whole cells and for measuring adenylcyclase activity in membranes are commercially available (see, e.g.,Gabriel et al. (2003). Assay Drug Dev. Technol. 1, pp. 291-303; Williams(2004). Nat. Rev. Drug Discov. 3, pp. 125-135, each incorporated byreference in their entireties), and are amenable for use in the devicesand methods provided herein. That is, cell populations in one or moremicrofluidic chambers can be assayed according to these methods.

Cisbio International (Codolet, France) has developed a sensitivehigh-throughput homogenous cAMP assay (HTRF, see Degorce et al. (2009).Current Chemical Genomics 3, pp. 22-32, the disclosure of which isincorporated by reference in its entirety) based on time resolvedfluorescence resonance energy transfer technology and can be used hereinto screen for an effector cell exhibiting an effect on a GPCR. Themethod is a competitive immunoassay between native cAMP produced bycells and a cAMP-labeled dye (cAMP-d2). The cAMP-d2 binding isvisualized by a MaB anti-cAMP labeled with Cryptate. The specific signal(i.e., energy transfer) is inversely proportional to the concentrationof cAMP in the sample, in this case, the amount of cAMP activated in areadout cell by an effector cell or an effector cell secretion product.As cAMP is being measured, readout cells are first lysed to free thecAMP for detection. This assay has been validated for bothG_(s)-(β₂-adrenergic, histamine H2, melanocortin MC4, CGRP and dopamineD₁) and G_(i/o)-coupled (histamine H₃) receptors.

cAMP assay kits based on fluorescence polarization are alsocommercially, e.g., from Perkin Elmer, Molecular Devices and GEHealthcare, and each is amenable for use as an effector cell assay inthe methods and devices provided herein. Accordingly, one embodiment ofthe present invention comprises selecting an effector cell and/or cellpopulation comprising one or more effector cells based on the result ofa cAMP fluorescence polarization assay. The method is used in oneembodiment to determine whether the effector cell activates (agonism) orinhibits (antagonism) on a particular GPCR.

In one embodiment, the AlphaScreen™ cAMP assay from Perkin Elmer, asensitive bead-based chemiluminescent assay requiring laser activation,is used in the devices provided herein to screen for an effector cellhaving an effect on a readout cell, specifically, the activation orinhibition of a GPCR.

DiscoveRx (world-wide-website: discoverx.com) offers a homogenoushigh-throughput cAMP assay kit called HitHunter™ based on a patentedenzyme (β-galactosidase) complementation technology using eitherfluorescent or luminescent substrates (Eglen and Singh (2003). CombChem. High Throughput Screen 6, pp. 381-387; Weber et al. (2004). AssayDrug Dev. Technol. 2, pp. 39-49; Englen (2005). Comb. Chem. HighThroughput Screen 8, pp. 311-318, each incorporated by reference intheir entireties). This assay can be implemented herein to detect aneffector cell having an extracellular effect or a variation in anextracellular effect on a readout cell expressing a GPCR.

Cellular events that result from GPCR receptor activation or inhibitioncan also be detected to determine an effector cell's property(ies)(e.g., an antibody producing cell's ability to activate or antagonize)on a readout cell. For example, in the case of the Gq coupled receptors,when the GPCR is activated, the Gq protein is activated, which resultsin the phospholipase C cleavage of membrane phospholipids. This cleavageresults in the generation of inositol triphosphates 3 (IP3). Free IP3binds to its target at the surface of the endoplasmic reticulum causinga release of calcium. The calcium activates specific calcium responsivetranscription vectors such as nuclear factor of activated T-cells(NFAT). Thus, by monitoring NFAT activity or expression, an indirectreadout of the GPCR in a readout cell is established. See. e.g.,Crabtree and Olson (2002). Cell 109, pp. S67-S79, incorporated byreference herein in its entirety.

Once activated more than 60% of all GPCRs are internalized. Utilizing atagged GPCR (typically done with a C-terminal GFP tag) the distributionof the receptor in one embodiment, is imaged in the presence and absenceof ligand. Upon ligand stimulation a normally evenly distributedreceptor will often appear as endocytosed puncta.

TABLE 4 GPCR functional assays for use with the present invention.Biological Reagents (accessory Assay measurements particles) BasisEndpoint Notes Europium- Membrane-based Europium-GTP Binding ofTime-resolved Proximal to GTP ™ GPCR europium- fluorescence receptorbinding (Perkin mediated labeled GTP to activation, Elmer) Guaninereceptor activated nonradioactive. nucleotide G proteins exchangeAlphaScreen ™ Cell-based cAMP MAb cAMP competes Luminescence Highsensitivity, (Perkin Elmer) cAMP conjugated with biotinyl-cAMPhomogeneous, accumulation acceptor bead, binding to high- amenable tostreptavidin-coated affinity streptavidin- automation, donor beads withcoated donor beads, broad chemoluminescence loss of signal due to linearcompound, reduced proximity of range of biotinyl-cAMP acceptor-donorbead detection Fluorescence Cell- or cAMP MAb, cAMP competes withFluorescence Homogeneous, polarization membrane-based fluorescentFluor-cAMP polarization amenable to (Perkin cAMP cAMP binding to cAMPautomation Elmer, accumulation MAb, loss of Molecular signal due toDevices, GE decrease in rotation Healthcare) and polarization HTRF cAMPCell-based, cAMP MAb cAMP competes Time-resolved Broad linear (Cisbio)cAMP conjugated with acceptor-labeled fluorescence range, accumulationwith eurocryptate, cAMP binding to high signal- acceptor moleculeeuropium-conjugated to-noise, labeled cAMP cAMP MAb, loss of homogenous,signal due to reduced amenable to europium-acceptor automation moleculeproximity HitHunter ™ Cell-based, cAMP MAb, ED- cAMP competes withFluorescence or Low compound (DiscoveRx) cAMP cAMP (enzyme ED-cAMP forluminescence interference, accumulation fragment dono- complementationof high cAMP conjugate) β-Gal activity with sensitivity, conjugatedpeptide, binding of acceptor homogeneous, acceptor protein, peptide,loss of amenable to lysis buffer signal as enzyme automationcomplementation is reduced IP₁ ™ (Cisbio) Cell-based IP₁ Europium- Lossof signal Time-resolved Homogeneous, accumulation conjugated IP₁ as IP₁competes fluorescence can be used for MAb, acceptor for binding ofconstitutively labeled IP₁ acceptor-labeled active Gq- IP₁ binding tocoupled GPCRs europium-MAb FLIPR ™ Cell-based, Caldium IncreasedFluorescence Sensitive, (Molecular increases in sensitive dye;fluorescence as homogeneous, Devices) intracellular caldium-3intracellular dye amenable to calcium binds calcium automationAequoScreen ™ Cell-based, Cell lines Calcium-sensitive LuminescenceSensitive, (EuroScreen) increases in expressing aequorin generateshomogeneous, intracellular select GPCRs a luminescent amenable tocalcium along with signal when a automation promiscuous coelenterazineor chimeric derivative G proteins and a is added mitochondriallytargeted version of apoaequorin Reporter gene Cell-based, Severalpromoter GPCR changes Fluorescence, Homogeneous, increases in plasmidsand in secondary luminescence, amplification reporter reportersmessengers alter absorbance of gene expression expression signal due toincreases of a selected in second reporter gene messengers activated byGPCR binding Melanophore Cell-based, Melanosomes Absorbance Sensitive,(Arena changes in aggregate homogeneous, Pharmaceuticals) pigment withinhibition no cell lysis, dispersion of PKA disperse amenable to withactivation of automation PKA or PKC Adapted from Thomsen et al. (2005).Current Opin. Biotechnol. 16, pp. 655-665, incorporated by referenceherein in its entirety for all purposes.

One embodiment of a work flow for a single cell antibody secreting cell(ASC)/antibody selection pipeline is shown in FIG. 1. In thisembodiment, a host animal is immunized with a target antigen and cellsare obtained from spleen, blood, lymph nodes and/or bone marrow one weekfollowing a final immunization boost. These samples are then optionallyenriched for ASCs by flow cytometry (e.g., FACS) or magnetic beadpurification using established surface markers (if available) or usingmicrofluidic enrichment. The resulting ASC enriched population is thenloaded into a microfluidic array of nanoliter volume chambers, with aloading concentration chosen to achieve from about one to about 500cells per chamber, or from about one to about 250 cells per chamber.Depending on the pre-enrichment step, individual chambers will comprisemultiple ASCs, single ASCs or zero ASCs. Chambers are then isolated byclosing microvalves and incubated to allow for antibodies to be secretedin the small chamber volume. Because ASCs typically secrete antibodiesat a rate of 1000 antibody molecules per second, and the volume ofindividual chambers provided herein are on the order of 2 nL, aconcentration of about 10 nM of each secreted monoclonal antibody isprovided in about 3 hours (each ASC secretes a unique monoclonalantibody). In a further embodiment, integrated microfluidic control isthen used for delivery and exchange of reagents in order to implementimage-based effector cell assays, which are read out using automatedmicroscopy and real-time image processing. Individual ASCs or cellpopulation comprising one or more ASCs that secrete antibodies withdesired properties (e.g., binding, specificity, affinity, function) arethen recovered from individual chambers. Further analysis of therecovered cell populations at limiting dilution is then carried out. Inthe case that an individual ASC is provided to a chamber and recovered,further analysis of the individual ASC can also be carried out. Forexample, in one embodiment, the further analysis includes single cellRT-PCR to amplify paired HV and LV for sequence analysis and cloninginto cell lines.

In another embodiment, after an animal is immunized and cells areobtained from spleen, blood, lymph nodes and/or bone marrow, the cellsmake up a starting population that are loaded directly into individualchambers of a microfluidic device provided herein, i.e., as a pluralityof cell populations, wherein individual cell populations are present ineach microfluidic chamber. An extracellular effect assay is then carriedout in the individual chambers on the individual cell populations todetermine if any of the individual cell populations comprise one or moreeffector cells responsible for an extracellular effect.

Although a host animal can be immunized with a target antigen prior tomicrofluidic analysis, the invention is not limited thereto. Forexample, in one embodiment, cells are obtained from spleen, blood, lymphnodes or bone marrow from a host (including human) followed by anenrichment for ASCs. Alternatively, no enrichment step takes place andcells are directly loaded into chambers of a device provided herein,i.e., as a plurality of cell populations, where individual cellpopulations are present in each chamber.

The methods provided herein allow for the selection of antibodies fromany host species. This provides two key advantages for the discovery oftherapeutic antibodies. First, the ability to work in species other thanmice and rats allows for the selection of mAbs to targets with highhomology to mouse proteins, as well as mAbs to human proteins thatcross-react with mice and can thus be used in easily accessiblepre-clinical mouse models. Second, mouse immunizations often result inresponses that feature immunodominance to a few epitopes, resulting in alow diversity of antibodies generated; expanding to other species thusgreatly increases the diversity of antibodies that recognize differentepitopes. Accordingly, in embodiments described herein, mice rats andrabbits are used for immunizations, followed by the selection of ASCsfrom these immunized animals. In one embodiment, a rabbit is immunizedwith an antigen, and ASCs from the immunized rabbit are selected forwith the methods and devices provided herein. As one of skill in the artwill recognize, rabbits offer advantages of a distinct mechanism ofaffinity maturation that uses gene conversion to yield greater antibodydiversity, larger physical size (more antibody diversity), and greaterevolutionary distance from humans (more recognized epitopes).

The immunization strategy, in one embodiment is a protein, cellular,and/or DNA immunization. For example, for PDGFRα, the extracellulardomain obtained from expression in a mammalian cell line, or purchasedfrom a commercial source (Calixar) is used to immunize an animal. ForCXCR4, in one embodiment, virus-like particle (VLP) preparations, ananoparticle having a high expression of GPCR in native conformation,from a commercial source (Integral Molecular) is used. Cell-basedimmunization is performed by overexpression of full-length proteins in acell line (e.g., 32D-PDGFRα cells for mice/rats, and a rabbit fibroblastcell line (SIRC cells) for rabbits; including protocols in which a newcell line is used in the final boost to enrich for specific mAbs. Avariety of established DNA immunization protocols are also amenable foruse with the present invention. DNA immunization has become the methodof choice for complex membrane proteins since it 1) eliminates the needfor protein expression and purification, 2) ensures native conformationof the antigen, 3) reduces the potential for non-specific immuneresponses to other cell membrane antigens, and 4) has been proveneffective for challenging targets (Bates et al. (2006). Biotechniques40, pp. 199-208; Chambers and Johnston (2003). Nat. Biotechnol. 21, pp.1088-1092; Nagata et al. (2003). J. Immunol. Methods 280, pp. 59-72;Chowdhury et al. (2001). J. Immunol Methods 249, pp. 147-154; Surman etal. (1998). J. Immunol. Methods 214, pp. 51-62; Leinonen et al. (2004).J. Immunol. Methods 289, pp. 157-167; Takatasuka et al. (2011). J.Pharmacol. and Toxicol. Methods 63, pp. 250-257, each incorporated byreference in their entireties for all purposes). All immunizations areperformed in accordance with animal care requirements and establishedprotocols.

Anti-PDGFRα antibodies have been previously produced in rats, mice, andrabbits, and comparison of the extracellular domain of PDGFRα showsseveral sites of substantial variation (FIG. 24). Thus it is expectedthat a good immune response is obtainable from this antigen. Anti-CXCR4mAbs have also been previously generated using both lipoparticles andDNA immunizations, so that this target is likely to yield a good immuneresponse. If needed, we will investigate using the co-expression ofGroEl or GM-CSF (either co-expressed or as a fusion) as a molecularadjuvant, as well as testing of different adjuvants and immunizationschedules (Takatsuka et al. (2011). J. Pharmacol. and Toxicol. Methods63, pp. 250-257 Fujimoto et al. (2012). J. Immunol. Methods 375, pp.243-251, incorporated by reference in their entireties for allpurposes).

The devices provided herein are based on Multilayer Soft Lithography(MSL) microfluidics (Unger et al. (2000). Science 7, pp. 113-116,incorporated by reference in its entirety). MSL is a fabrication methodthat provides for increased sensitivity through small volume reactions;high scalability and parallelization; robust cell culture; flexibilityand fluid handling control needed for complex assays; and greatlyreduced cost and reagent consumption.

The number of effector cells isolated per device run (i.e., number ofcells in each chamber of a device) is a function of the concentration ofcells in a cell suspension loaded onto a device, the frequency in thecell suspension of the specific effector cells being selected for, andthe total number of chambers on a device. Devices with arrays up to andgreater than 40,000 effector cell assay chambers are contemplated.

Amongst all microfluidics technologies, MSL is unique in its rapid andinexpensive prototyping of devices having thousands of integratedmicrovalves (Thorsen et el. (2002). Science 298, pp. 58-584,incorporated by reference in its entirety). These valves can be used tobuild higher-level fluidic components including mixers, peristalticpumps (Unger et al. (2000). Science 7, pp. 113-116) and fluidicmultiplexing structures (Thorsen et el. (2002). Science 298, pp. 58-584;Hansen and Quake (2003). Curr. Opin. Struc. Biol. 13, pp. 538-544,incorporated by reference in their entireties herein) thus enabling highlevels of integration and on-chip liquid handling (Hansen et al. (2004).Proc. Natl. Acad. Sci. U.S.A. 101, pp. 14431-1436; Maerkl and Quake(2007). Science 315, pp. 233-237, each incorporated by reference intheir entireties). (FIG. 25).

FIG. 25A shows an optical micrograph of a valve made by MSL. Twocrossing microfabricated channels, one “flow channel” for the activefluids (vertical) and one control channel for valve actuation(horizontal), create a valve structure. The flow channel is separatedfrom the control channels by a thin elastomeric membrane to create a“pinch valve.” Pressurization of the control channel deflects themembrane to close off the flow channel. FIG. 25B shows a section of anMSL device integrating multiple valves (filled with green and blue fooddye). FIG. 25C is a section of a device having a total of 16,000 valves,4000 chambers, and over 3000 layer-layer interconnects (arrow). FIG. 25Dshows an example of a microfluidic device with penny for scale. Devicesshown are for illustration of the MSL fabrication technology.

The assay chambers of a device, in one embodiment has an average volumeof from about 100 μL to about 100 nL. For example, in one embodiment,one or more properties of an effector cell is assayed within amicrofluidic chamber comprising a cell population wherein the volume ofthe microfluidic chamber is about 100 μL, about 200 μL, about 300 μL,about 400 μL, about 500 μL, about 600 μL, about 700 μL, about 800 μL,about 900 μL or about 1 nL. In another embodiment, the volume of themicrofluidic chamber is about 2 nL. In another embodiment, the volume ofthe microfluidic chamber for assaying a property of an effector cell ina cell population is from about 100 μL to about 100 nL, from about 100μL to about 50 nL, from about 100 μL to about 10 nL, from about 100 μLto about 1 nL, from about 50 μL to about 100 nL, from about 50 μL toabout 50 nL, from about 50 μL to about 10 nL or from about 50 μL toabout 1 nL. In even another embodiment, the volume of the microfluidicchamber for assaying a property of an effector cell in a cell populationis about 10 nL, about 20 nL, about 30 nL, about 40 nL, about 50 nL,about 60 nL, about 70 nL, about 80 nL, about 90 nL or about 100 nL.

The MSL fabrication process takes advantage of well-establishedphotolithography techniques and advances in microelectronic fabricationtechnology. The first step in MSL is to draw a design of flow andcontrol channels using computer drafting software, which is then printedon high-resolution masks. Silicon (Si) wafers covered in photoresist areexposed to ultraviolet light, which is filtered out in certain regionsby the mask. Depending on whether the photoresist is negative orpositive, either areas exposed (negative) or not (positive) crosslinksand the resist will polymerize. The unpolymerized resist is soluble in adeveloper solution and is subsequently washed away. By combiningdifferent photoresists and spin coating at different speeds, siliconwafers are patterned with a variety of different shapes and heights,defining various channels and chambers. The wafers are then used asmolds to transfer the patterns to polydimethylsiloxane (PDMS). In oneembodiment, prior to molding with PDMS and after defining photoresistlayers, molds are parylene coated (chemical vapor depositedpoly(p-xylylene) polymers barrier) to reduce sticking of PDMS duringmolding, enhance mold durability and enable replication of smallfeatures

In MSL, stacking different layers of PDMS cast from different molds ontop of each other is used to create channels in overlapping “flow” and“control” layers. The two (or more) layers are bound together by mixinga potting prepolymer component and a hardener component at complementarystoichiometric ratios to achieve vulcanization. In order to create asimple microfluidic chip, a “thick” layer (e.g., between from about200-2000 pins) is cast from the mold containing the flow layer, and the“thin” layer (e.g., between from about 25 to about 300 μms) is cast fromthe mold containing the control layer. After partial vulcanization ofboth layers, the flow layer is peeled off its mold, and aligned to thecontrol layer (while still present on its mold, by visual inspection.The control and flow layers are allowed to bond, for example at 80° C.for about 15-60 minutes. The double slab is then peeled from the controlmold, and inlet and outlet holes are punched and the double slab isbonded to a blank layer of PDMS (i.e., a flat layer of PDMS with nostructural features). After allowing more time to bond, the completeddevice is mounted on a glass slide. Fluid flow in the device iscontrolled using off-chip computer programmable solenoids which actuatethe pressure applied to fluid in the channels of the control layer. Whenpressure is applied to these control channels, the flexible membranebetween the overlapping orthogonal control and flow lines deflects intothe flow channel, effectively valving the flow. Different combinationsof these valves can be used to create peristaltic pumps, multiplexercontrols and isolate different regions of the chip

With respect to the flow layer, assay chambers and channels forcontrolling fluidic flow to and from the assay chambers are defined bythe photoresist layers. As will be appreciated by one of skill in theart, the thickness of a photoresist layer can be controlled in part bythe speed of spin coating and the particular photoresist selected foruse. The bulk of the assay chambers, in one embodiment, are defined byan SU-8 100 feature which sits directly on the Si wafer. As known tothose of skill in the art, SU-8 is a commonly used epoxy-based negativephotoresist. Alternatively, other photoresists known to those of skillin the art can be used to define assay chambers with the heightsdescribed above. In some embodiments, the assay chambers have a heightand width of 50-500 μM and 50-500 μM, respectively, as defined by theSU-8 features.

MSL fabrication techniques allow for a wide range of device densities,and chamber volumes to be fabricated. For the devices provided herein,in one embodiment, from about 2000 to about 10,000 effector cellanalysis chambers are provided in a single integrated device. Theeffector cell analysis chambers, in one embodiment, have an averagevolume of from about 1 nL to about 4 nL, for example, from about 1 nL toabout 3 nL, or from about 2 nL to about 4 nL. The effector cell analysischambers, in one embodiment, are connected in a serial format, asdepicted in FIG. 26. By way of illustration, a device with 4032individual analysis chambers (average volume of 2.25 nL) connected inserial format achieve a screening throughput of approximately 100,000cells per run (FIG. 8). The integrated microfluidic valves harnessed inthe devices provided herein allow for chamber isolation, andprogrammable washing with reagents selected from a plurality of inlets,for example from 2 to about 32 inlets, 2 to about 20 inlets, 2 to about15 inlets, 2 to about 10 inlets, or from 2 to about 9 inlets, or from 2to about 8 inlets, or from 2 to about 7 inlets or from 2 to about 6inlets. Additional inlets are provided to control valve pressure (FIG.26).

The devices provided herein harness a gravity-based immobilization ofcells and/or particles. Gravity based immobilization allows forperfusion of non-adherent cell types, and in general, buffer and reagentexchange within chambers. Each chamber has a cubic geometry with anaccess channel passing over the top (FIG. 26). For example, a chamberprovided herein, in one embodiment, has the following dimensions: 50-250μm×50-250 μm×50-250 μm; l×w×h, e.g., 150 μm×100 μm×150 μm; l×w×h. Duringloading, particles (e.g., cells or beads) follow streamlines and passover tops of chambers, but fall to the bottom of the chambers when theflow is stopped. Due to the laminar flow profile, the flow velocity isnegligible near the chamber bottom. This allows for perfusion of thechamber array, and the exchange of reagents via combinedconvection/diffusion, without disturbing the location of non-adherentcells (or beads) in the chambers.

Importantly, the devices provided herein allow for the long term cultureand maintenance of cells, whether effector, accessory or of the readoutvariety. Microfluidic arrays of chambers are fabricated within a thickmembrane (e.g., from about 150 μm to about 500 μm thick, about 200 μmthick, about 300 μm thick, about 400 μm thick or about 500 μm thick) ofPDMS elastomer that is overlaid a reservoir of medium, for example 1 mLof medium as described previously (Lecault et al. (2011). Nature Methods8, pp. 581-586, incorporated by reference herein in its entirety for allpurposes). The proximity of the medium reservoir (osmotic bath) to thecell chambers effectively blocks evaporation (through the gas-permeablePDMS material) and ensures robust cell viability and where cells are notfully differentiated, growth over several days, and is critical forachieving long-term culture in nL volumes with growth rates and cellularresponses that are identical to μL volume formats. FIG. 27 shows aschematic of the layers of the devices used herein.

The membrane design of the devices provided herein also enables theselective recovery of cells from any chamber by piercing the uppermembrane with a microcapillary.

The device architectures provided herein are designed so that thesoluble secretion products of effector cells are not washed away from achamber when additional components, e.g., accessory particles or cellsignaling ligands are added to the chamber. Additionally, the devicesprovided herein allow for the addition of components to a chamberwithout the introduction of cross-contamination of secretion products(e.g., antibodies) between individual chambers of a device. Inembodiments where chambers are connected in serial, medium exchangerequires flushing the entire array. Flushing the entire array, in oneembodiment, results in the loss of antibody from each chamber andintroduces cross-contamination to downstream chambers. However, wheresecretion products bind to a surface of a chamber or a readout particlebound to a surface, cross-contamination and/or loss of secretionproducts is not a significant problem, as secretion products areimmobilized.

In one embodiment, cross-contamination and/or loss of secretion productis minimized by using an “inflatable chamber” design as shown in FIG.28A-D. Each chamber has a single inlet, and are connected to a commonchannel through a short “access channel” controlled by a microvalve. Thetop of each chamber is overlaid by a recess, separated from the chamberby a thin (˜10 pin) membrane, thus allowing for significant volumeexpansion when the chamber is pressurized. In one embodiment, chambersare inflatable to 2× volume, e.g., for 2 nL to 4 nL or 1 nL to 2 nL or2.5 nL to 5 nL. In another embodiment, chambers are inflatable to 1.5×volume, e.g., for 2 nL to about 3.5 nL by application. Particles (cellsor beads), in one embodiment, are loaded into inflatable chambers byinflating the chamber, allowing the particle to settle under gravity,and then deflating chamber. Similarly, a process of sequentialinflation, diffusive mixing, and deflation, allows for washing of thechamber contents and/or exchange of medium. This approach enables theaddition of soluble ligand without losing secretion products, as theyare only diluted by less than 50%. Inflatable chambers also eliminatethe potential for cross-contamination since the chambers are notconnected in serial. The simplicity of this architecture allows for theintegration of dense arrays, and in one embodiment, is applicable indevices having 10,000 chambers on an area of just over one square inch.

In one embodiment, the microfluidic devices provided herein are operatedin flow conditions that suppress inertial effects and are dominated byviscosity. This fluid flow regime is characterized by a low Reynoldsnumber Re=ρdu/η, where ρ is the density of the fluid, d is thecharacteristic length scale of the channel, u is the characteristicvelocity of the flow, and η is the viscosity of the fluid. At low Re thefluid flow streamlines become predictable and can be designed for thedesired effect. Referring to FIGS. 29 and 30, for instance, if the goalis to load effector cells 40 only on the left side of the chamber 41,the device in one embodiment, is designed with a restriction upstream,e.g., formed by deflector 42 in FIG. 30, of the chamber thatpreferentially directs effector cells to the left side of the inletchannel 43. As effector cells 40 enter into the chamber 41 under flow,they continue into the left side of the chamber, following thestreamlines of the flow, provided that the time of transport is chosensuch that the effector particles do not diffuse substantially acrossstreamlines. Similarly, readout particles, in one embodiment, aredirected to the right of the chamber by use of an auxiliary channelconnected to the inlet channel that results in the positioning of thereadout particles in the right side of the inlet channel. In anotherembodiment, the readout particles are introduced through a differentchannel that accesses the chamber, including the outlet channel. It willbe understood by those of skill in the art that the design of laminarflow profiles provides great flexibility in the direction of particlesto a specific region of the chamber, if specific placement within achamber is desired.

Segregation of effector cells from readout particles in a particularchamber can also be achieved via the use of a structural element, bymanipulating the flow within a flow channel or channels of the device,stochastic loading, a magnetic field, an electric field or dielectricfield, a gravitational field, a modification to a surface of themicrofluidic chamber that affects adhesion, and relative buoyancy of theeffector cells and the readout particles, or a combination thereof.

In one embodiment, effector cells and/or readout cells are confined to achamber or a portion of a chamber (e.g., an effector zone or readoutzone) using a structural element within the chamber.

“Effector zone” as used herein, is a region of a microfluidic chamber inwhich an effector cell, a population of effector cells (or subpopulationthereof), is retained.

“Readout zone” as used herein, is a region of a microfluidic chamber inwhich a readout particle is kept segregated from effector cells, and inwhich a functionality of the effector cell(s) may be detected. Forexample, when an “effector zone” and a “readout zone” are threedimensional regions of a microfluidic device, they may be individualchambers (for example, a compound chamber), where the chambers are influid communication with one another.

As described herein, in one embodiment, one or more structural elementsare used for the distribution of effector cells and/or readout particleswithin one or more chambers. In a further embodiment, the one or morestructural elements are used for the retaining of effector cells and/orreadout particles within defined regions of a chamber, e.g., an effectorzone and/or readout zone. In some embodiments, the use of suchstructural elements is coupled with the use of a field (e.g., gravity,dielectric, magnetic, etc.) to achieve a retaining function. Forexample, in one embodiment, a cell fence is utilized to trap cells(effector or readout) at a certain chamber location. Cell fences havebeen described previously in PCT Application Publication No.2012/162779, and the disclosure of which is incorporated by reference inits entirety for all purposes. “Cell fence,” as used herein, refers toany structure which functions to restrict the movement of cells andreadout particles, but which may permit the movement of other cellproducts, within a microfluidic chamber.

In embodiments where cell fences are utilized, each fence is defined bya thinner SU-8 2010 feature which sits on top of the SU-8 100 feature.Such a structure is depicted schematically in FIG. 31. Fabrication ofcell fences have been described previously in PCT ApplicationPublication No. 2012/162779, the disclosure of which is incorporated byreference in its entirety. In one embodiment where a cell fence isutilized to capture an effector cell or a population of cells optionallycomprising one or more effector cells, microfluidic chambers are definedusing SU-8 100 negative photoresist (typically 160 μm in height)following standard protocols, except that the final step, development isomitted. Specifically, a Si wafer spun with SU-8 100 photoresist iscooled after the post-exposure bake, and instead of developing thephotoresist, SU-8 2010 is spun on top of the undeveloped SU-8 100,typically at a height 10-20 um. The thickness of the SU-8 2010determines the height of the fence. The depth of each chamber was thecombined height of both photoresist layers.

In alternative cell fence embodiments, the SU-8 100 layer is fullydeveloped, and the wafer is coated with an additional layer of SU-8 100which is higher than the first layer, the difference becoming the heightof the fence.

Structural elements described herein may provide for the retention ofeffector cells and readout particles on different sides of a chamber byuse of gravity; in this case gravity is directed towards the floor ofthe chamber and provides a force that impedes the cell from rising overthe fence. It should be noted that microscopic particles may be subjectto Brownian motion that may cause them to “diffuse” over a fence in astochastic manner. However, the probability that a particle willspontaneously rise over the fence by Brownian motion is determined bythe Boltzmann distribution and is proportional to the Boltzmann factorwhere E is the potential energy of rising the particle to a height equalto the height of the fence (E=volume of particle×the difference betweenthe density of the particle and the density of the liquid×gravity×theheight of the fence), K is the Boltzmann constant, and T is thetemperature of the liquid. Thus, one of skill in the art can design andfabricate the height of a fence such that the probability of a particlespontaneously “diffusing” over the fence is negligibly small. It willalso be appreciated by one of skill in the art that a fence of a givenheight presents a barrier to some particles (e.g., beads or cells),depending on the potential energy required to pass over the fence, butwill not present a barrier to particles having appropriately lowapparent mass in the liquid. For example, a fence having a height ofabout 20 μm does not allow for a cell to spontaneously pass bydiffusion, but presents essentially no barrier to the diffusion ofproteins. In one embodiment, gravity used in conjunction with astructural element for retaining cells. However, other forces such asmagnetic gradient forces, dielectric forces, centrifugal forces, flowforces, or optical forces, may be similarly used.

Structural elements may also be effective in retaining effector cells orreadout particles by providing a mechanical barrier to passage. Forinstance, the use of a fence that reaches to within a gap of the roof ofa chamber, where ‘d’ is smaller than the diameter of all effector cellsor readout particles, acts as a barrier without the need for applicationof another force. Such a structure may also be designed to selectivelypartition effector cells or particles on different sides of a chamber bydesigning the gap to allow for one type of particle to pass but not theother. Referring to FIG. 32, for example, in one embodiment, effectorcells 120 have a diameter of 10 microns, and a mixture of the effectorcells and readout particles 121 (having a diameter of 1 micron) areloaded into one side of a chamber that is separated from the other sideby a fence 122 having a 5 micron gap (as measured to the roof of thechamber). By tipping the device appropriately, the 1 micron readoutparticles 121 are transferred to the other side of the chamber, whileleaving the effector cells 120 on the other side. In such a manner, theeffector cells are positioned in the effector zone of a chamber whilethe readout particles are partitioned in the readout zone.

Wells within a chamber can also be used to sequester assay reagents,effector cells and/or readout cells. For example, representative devicegeometries are shown in FIGS. 33 and 34. In one embodiment, a chamber isdesigned with an array of wells, whereby small wells are defined at thebottom of a chamber (FIG. 33). Wells may be any shape, and are designedbased on the effector cell or readout particle they are designed toassociate with. Both square and circular shaped wells are amenable foruse with the devices described herein, and generally, any polygon shapedwell is amenable for use in a chamber. Various prototypes werefabricated with square and circular wells, as well as square andcircular ‘posts.’ Effector cells and readout particles are loaded intothe device at concentration such that each well within a chambercontains a subpopulation of cells or readout particles. For example, inone embodiment, each well within a chamber is addressed to contain oneor zero effector cells. Such a design can spatially constrain theparticles/cells. In embodiments where discrete “effector zones” and“readout zones” are utilized, an effector cell defines the well in whichit resides as an “effector zone” and a readout particle defines the wellin which it resides as a “readout zone”.

FIG. 34 shows a “bead trap” embodiment that can be used in the devicesof the present invention. This design spatially confines a plurality ofreadout particles (e.g., a plurality of beads or readout cells) or cellpopulation to a specific spatial position within a chamber. Such adesign overcomes problems that can be associated with beads or particlesthat otherwise move during an assay, thereby simplifying downstreamimaging and image analysis. Having a fixed position for the readoutparticle is also advantages due to the diffusion distance betweeneffector cell and readout particle being better controlled.

The operation of a bead trap (shown in FIG. 34), in one embodiment,occurs as follows: readout particles are loaded into a chamber and thenallowed to settle by gravity to the bottom of the chambers, while thechamber is tipped toward the upper right corner (i.e., the zone enclosedby the perpendicular cell fences and generally circular particle trapopening toward the upper right quadrant). After the readout particle orparticles have settled to the bottom of the chamber, the device could betipped in the opposite direction (along the same axis) so that thereadout particle or particles slide or roll along the bottom of thechamber and into the circular ‘trap’ feature in the center of thechamber. As mentioned above the same method and device may be employedto sequester effector cells.

In one embodiment, effector cells and readout particles are positionedwithin a chamber into an effector zone and readout zone usingmicro-fabricated structures that are designed to retain one or moretypes of particles (e.g., cells). For example, in one embodiment, theflow of effector cells or readout particles may be designed to intersectwith micro-fabricated cup structures that are designed to retainparticles but which allow the flow to pass through. Such structures canbe designed such that they can accommodate only a fixed number ofparticles or cells, or a defined size range of particles or cells,making the structures selective for different particle types. Such trapsmay be positioned substantially in the chamber or at the inlet andoutlets of the chambers.

In another embodiment, referring to FIG. 35, effector cells 81 areprovided at the bottom of a chamber using gravitation, while readoutparticles 82 are positioned at the outlet 83 of the chamber using a trapstructure 84 that is fabricated in the outlet channel.

According to one embodiment of the invention, one or more recesses (alsoreferred to herein as “cups”) at the bottom of a chamber are provided tosegregate effector cells from readout particles. Referring to FIG. 36,for example, dead-end cups 361 are provided at the bottom of a chamber362 which have a width smaller than the average diameter of the effectorcell 363 but greater than the diameter of the readout particle 364. Inthis configuration, readout particles 364 sink to the bottom of the cups361 while effector cells 363 are retained above the cup entrance. Insome embodiments, the bottom of the cup 361 is accessed through a porousmembrane and channel structures beneath, to augment fluidic access tothe readout particles in dead-end cups that are covered with effectorcells.

In one embodiment, structural elements are positioned in a flow channelor chamber to retain one or more particles or cells. Referring to FIG.37, structural elements 371 (or functionalized surface patches, etc) maybe positioned in the flow channel (not necessarily a microwell) toretain one or more effector cells 372 and/or one or more readoutparticles 373. For example, trap structures may be designed to retainparticles or cells with specific physical properties (e.g. size) or maybe non-specific (random distribution of effector cell and readoutparticle). Valves 374 can be used to separate adjacent chambers.

In an extension of the design described above (e.g., FIG. 37),structural elements in the flow channel are designed to retain effectorcells and readout particles in close proximity to one another. Referringto FIG. 38, in one embodiment, one unit includes an effector cell trap381 to trap an effector cell 382, and microstructure 383 to retain oneor more readout particles 384. Readout particles with a diameter smallerthan the gap of the cell trap pass the cell trap and are retained in themicrostructure 383 located downstream of effector cell trap 331.Effector cells with a diameter larger than gap 385 in the effector celltrap 381 are retained, as discussed above. In another embodiment,microstructures are designed so that the distance and location ofdifferent types of readout particles (or effector cells) are welldefined.

In addition to barriers such as fences between effector cells andreadout particles, porous membranes may also function as a barrier andtherefore, are amenable for use with the present invention. For example,referring to FIG. 39, in one embodiment, a porous membrane 391 (alsoreferred to as a diffusion channel) is fabricated between effector cellchambers 392 holding effector cells 395 and readout particle chambers393 holding readout particles 394, providing a horizontal arrangement.In such horizontal arrangements, the porous membrane 391, in oneembodiment, is substituted by a sieve valve or a fluidic channel with across-section smaller than the diameter of the effector cells or readoutparticles. Sieve valves have previously been described in U.S. PatentApplication Publication No. 2008/0264863, the disclosure of which isincorporated by reference in its entirety for all purposes.

In another embodiment, a porous membrane is fabricated between PDMSlayers to provide a vertical arrangement. In this embodiment, aneffector zone is provided in one PDMS layer and the readout zone isprovided in the second PDMS layer. One advantage of a verticalarrangement is that the spacing between the effector zone and thereadout zone is well-defined.

Porous membranes incorporated within PDMS devices have been reportedpreviously, for example, by Aran et al. (2010). Lab Chip 10, pp.548-552; Cheuh et al. (2007). Anal. Chem. 79, pp. 3504-3508, thedisclosure of which is incorporated by reference in its entirety hereinfor all purposes. In one embodiment, a porous membrane is fabricatedaccording to one of the following embodiments. In another embodiment, aPDMS layer is made porous by adding an immiscible fluid to the uncuredcomponents of the PDMS. During the baking step, the immiscible fluidevaporates. In yet another embodiment, a PDMS membrane is perforatedfollowing curing using laser ablation or another removal technique. Ineven another embodiment, a PDMS membrane is cast on one or moremicrostructures with a height exceeding the thickness of the membrane inorder to make the PDMS membrane porous.

In one embodiment, a structure that retains one or more effector cellsand/or readout particles is a temporary structure and/or removable. Inone embodiment, as illustrated in FIG. 40, readout particles 401 (e.g.,beads) are stacked against a sieve valve 402 (a valve that blocks onlypart of the channel cross-section). A layer of non-functional beads 403are then stacked against the readout particles 401, providing a barrier.A layer of effector cells 404 are then provided, and stacked against thelayer of non-functional beads 403. As will be understood from FIG. 40,effector cells 404, in one embodiment, are provided through the feedchannel 405. Effector cell assays measuring the effect of one or moreeffector cells 404 on one or more readout particles 401, in oneembodiment, are carried out using the lower bus channel 406. Oneadvantage of temporary structures is that chamber contents arerecoverable, and in some embodiments, selectively recoverable, throughthe lower bus channel by opening one or multiple of the sieve valves.

In addition to microfabricated retention methods or fields, hydrogelssuch as agarose are amenable for use in order to localize effector cellsand/or readout particles. For example, in one embodiment, readoutparticles and/or effector cells can be loaded in liquid agarose, forexample, photosensitive agaraose, which solidifies on-chip. As such, themovement of effector cells or readout particles is restricting, whichsimplifies the imaging process while still allowing diffusive transport.Upon re-melting of the agarose, in one embodiment, selective remelting,effector cells are recovered using microfluidic methods discussedherein. In another embodiment, immobilized cells are selectivelyrecovered using a micromanipulator or a robotic method.

In one embodiment, segregation of effector cells and readout particlesin a particular zone within a chamber, or a particular chamber, in oneembodiment, is achieved by a specific flow profile, stochastic loading,a magnetic field, an electric field or dielectric field, a gravitationalfield, a modification to a surface of the microfluidic chamber, andselecting a particular relative buoyancy of the effector cells and thereadout particles, or a combination thereof. For example, in oneembodiment, either the cell population is labeled with magneticparticles or the readout particles are magnetic, such that provision ofa magnetic field to the top surface of a particular chamber draws thecell population or readout particles upward, and away from either thereadout particles, in the case that the cells are magnetically labeled,or the cell population, in the case where readout particles aremagnetically labeled. In another embodiment, the specific gravity of thefluid in which the cell population and readout particles are incubatedcan be chosen to facilitate separation of the effector cells and readoutparticles based on their relative buoyancy. Certain particle and cellsequestration methods are discussed in greater detail below.

Effector cells and readout particles, in one embodiment, are distributedwithin a chamber by functionalizing one or more walls of the chamber. Inone embodiment, the surface functionalization is performed by graft,covalently linking, adsorbing or otherwise attaching one or moremolecules to the surface of the chamber, or modifying the surface of thechamber, such that the adherence of cells or particles to the chambersurface is altered. Nonexclusive examples of such functionalizations foruse herein are the non-specific adsorption of proteins, the chemicalcoupling of proteins, the non-specific adsorption of polymers, theelectrostatic adsorption of polymers, the chemical coupling of smallmolecules, the chemical coupling of nucleic acids, the oxidization ofsurfaces, etc. PDMS surface functionalization has been describedpreviously, and these methods can be used herein to functionalizesurfaces of the devices provided herein (see, e.g., Zhou et al. (2010).Electrophoresis 31, pp. 2-16, incorporated by reference herein for allpurposes). Surface functionalizations described herein, in oneembodiment, selectively bind one type of effector cell (e.g., aneffector cell present in a cell population), or selectively bind onetype of readout particle. In another embodiment, the surfacefunctionalization is used to sequester all readout particles present ina chamber.

In yet another embodiment, a surface functionalization or a plurality ofdifferent surface functionalizations are spatially defined within achamber of a device. Alternatively, a surface functionalization orplurality of surface functionalizations covers the entire chamber. Bothof these embodiments are useful for the distribution of effector cellsare readout particles into distinct locations within a microfluidicchamber. For instance, in embodiments where the entire chamber isfunctionalized with a molecule that binds all types of introducedreadout particles, the particles are directed to different regions ofthe device, using the methods described above, where they becomeimmobilized on the surface. In another embodiment, the entire chambermay be functionalized to bind only one specific type of readoutparticle. In this case, all particles, in one embodiment, are firstdirected to one region using one of the methods described herein,causing a subset of particles to adhere to the chamber surface in thefunctionalized region, followed by exerting a force towards a differentregion that displaces only the particles that do not bind thefunctionalized surface or surfaces. In yet another embodiment, regionsof the device (e.g., different chambers or regions within a singlechamber) are functionalized with different molecules that selectivelybind different subsets of effector cells and/or readout particles, suchthat inducing the interaction of the effector cells and/or readoutparticles with substantially the entire chamber surface results in thepartitioning of different particle or cell types in different regions.As described herein, it is intended that surface functionalization maybe used in isolation or in combination with the other methods describedherein for effector cell and readout particle manipulation. Multiplecombinations of particle and cell sequestration methods, together withmultiple fluidic geometries are possible.

In another embodiment, effector cells and/or readout particles arepositioned within a chamber by the use of a magnetic field. It will beunderstood that to manipulate and position an effector cell(s) and/or areadout cell(s) magnetically, the effector cell(s) and/or a readoutcell(s) are first functionalized with or exposed to magnetic particlesthat bind to them. In one embodiment, the magnetic field is externallycreated, i.e., by the use of a magnet outside of the microfluidicdevice, using a permanent magnetic, an electromagnet, a solenoid coil,or of other means. In another embodiment, referring to FIG. 41, themagnetic field is generated locally by a magnetic structure 90integrated into, or separate from, the device. The magnetic field, inone embodiment is applied at different times, and in one embodiment, themagnetic field is applied in conjunction with particle loading, toinfluence the position of effector cells and/or readout particles thatrespond to a magnetic field.

It will be appreciated by those skilled in the art that commerciallyavailable beads or nanoparticles that are used in the separation and orpurification of biological samples can be used in the devices andmethods provided herein. For example, “Dynabeads” (Life Technologies)are superparamagnetic, monosized and spherical polymer particles, and inone embodiment, are used in the devices and methods provided herein asreadout particles. Magnetic particles conjugated with molecules thatspecifically bind different target epitopes or cell types are well-knownin the art, and are also amenable for use with the devices and methodsprovided herein. When in the presence of a magnetic field having anon-uniform property, such magnetic particles are subjected to a forcethat is directed towards the gradient of the magnetic field. Thisgradient force, in one embodiment, is applied to position particleswithin a chamber.

Referring to the embodiment depicted in FIG. 42, it will be appreciatedthat a gradient force may be used to specifically apply a force to asubset of particles in order to preferentially direct one subtype, i.e.,particles 100, within a chamber. In one embodiment, this gradient forceis applied before particles enter the chamber in order to position themat a given position of the inlet channel, thereby resulting in particleloading to a specific region of the chamber. In another embodiment, thegradient force is applied prior to, during, or after the loading ofparticles into the chambers.

In addition to flow forces, a cell or particle (e.g., effector particleor readout particle) in the flow may be subject to “body forces” thatare derived from the action of an external field. The orientation of themicrofluidic devices provided herein, within a gravitational field maybe used to direct effector cells and readout particles to a specificregion of a device chamber. As illustrated in FIG. 43, one such methodinvolves physically tilting the entire microfluidic device and sampleholder along the axis of a fence, and waiting for a sufficient timeperiod for the respective particles to settle by gravity to either sideof the fence. In one embodiment, the tilt angle is from about 30 toabout 50 degrees, for example from about 30 degrees to about 45 degrees.However, it should be understood that this angle can be adjusteddepending on the cell or particle loaded, the flow rate, and theviscosity of the solution. It will be appreciated that the tipping of adevice in different orientations may be used to direct multiple sets ofparticles to multiple distinct regions, depending on the timing of thetipping and the introduction of the particles.

A person skilled in the art will understand that the use of gravity todirect particles within a chamber can be accomplished in multiple steps.Referring to FIG. 44, by way of example, a device is turnedsubstantially upside down, and the particles 51 are directed to one sideof a chamber roof by tipping the device. Next, the device is rotatedback to the upright position quickly, such that the particles do notmove far during the process of returning the device to the uprightposition. The particles then settle to the floor of the chamber, butremain on the right side of the chamber due to the presence of fence 50.In this way, features on the roof of the chamber can be used tosegregate particle types, followed by transfer of the particles to thefloor of the respective chamber without substantially changing thelateral positioning of the particles within the chamber. It will beunderstood by those in the art that the time for flipping the device inorder to localize particles as described above is dependent in part onthe velocity at which the particles fall through the liquid and themaximum displacement that can be accepted for accurate positioning. Thevelocity of a particle falling through a fluid is in the direction ofthe gravitational field and can be calculated according to the followingequation, which is known to those of skill in the art:

U=V _(particle)*(ρ_(particle)−ρ_(liquid))*g*γ ⁻¹

Where V_(particle) defines the volume of the particle, g is theacceleration of gravity, and γ is the drag coefficient of the particle.For a spherical particle, γ is well approximated by the Stokes dragequation as:

γ_(sphere)=6πηr

In some embodiments of the invention where gravity is used to positionparticles within a chamber, the effective size and/or density of theparticles are manipulated by using smaller particles that bind on theparticles surface. For example, the effector cell may be exposed toferromagnetic microbeads that are functionalized to bind the effectorcell, thereby causing the effector cell to have a much higher effectivedensity and to have a slightly larger drag coefficient, the net effectof which is to make the cell fall faster through the gravitationalfield.

Also within the scope of the invention, and related to the use ofgravity for the positioning of particles, is the use of buoyancy of theparticles within the fluid in order to direct fluid flow and effectorcell and readout particle positioning. Referring to FIG. 45, forexample, in one embodiment, effector cell 74 and readout particle 73have different densities, such that the density of effector cell 74 isgreater than that of the liquid in the chamber 77, and the density ofthe readout particle is less than that of the liquid, and the device isplaced within a gravitational field such that the gravitational field isdirected downwards in the chamber from the roof 75 to the floor 76, theeffector cell and the readout particle are partitioned to the floor andthe roof of the device, respectively, thereby defining the effector andreadout regions of the chamber 77. In one embodiment, this effect iscontrolled by the addition of components to the liquid that change thedensity and/or by modification of the particles, or selection of thereadout particle 73 and effector cell 74, such that they have theappropriate density differences. It should be noted that by exchangingmedium in the chamber the partitioning of particles may be reversiblymodulated. Such an exchange is within the scope of the presentinvention.

Effector cells and/or readout particles, in one embodiment, arepartitioned to different regions of a chamber by the use of anelectrostatic field and/or dielectric field that impart a force to theparticles. These fields, in one embodiment, are generated externally tothe device. In another embodiment, the electrostatic and/or dielectricfield is generated locally using one or more micro-fabricated electrodeswithin the microfluidic device. Such electrodes can be defined in ametal film on the substrate the device is mounted on, integrated into aseparate layer of the device, or defined by filling microfluidicchannels with a conducting liquid. There are many examples of theintegration of electrodes within microfluidic devices and variousgeometries and methods for integration will be apparent to those skilledin the art. For example, the electrodes disclosed by Li et al. (2006).Nano Letters 6, pp. 815-819, incorporated by reference herein in itsentirety), are amenable for use with the present invention.

In one embodiment, a dielectric field is applied to one of the devicesdescribed herein, and the dielectric field is designed with a frequencythat results in a differential force on particles having differentialproperties, which results in the separation of the particles bydifferential properties. An example of using dielectric fields toposition effector cells and readout particles within a chamber using adielectric field produced by an integrated electrode is shown in FIG.46. In this embodiment, effector cells 110 are loaded into a chamberhaving a fence structure 111 at the bottom, through an inlet channel 112situated on the top of the device. On loading the effector cells 110, anelectrode 113 is used to generate a dielectric field with a gradientthat results in a force on the effector cells 110. This force, directedtowards one side of the chamber, causes the effector cells 110 toselectively fall (under the influence of gravity) into the effectorregion of the chamber. Repeating this process with readout particles, anelectrode on the other side (readout zone) of the chamber may then beused to load the readout particles into the readout zone. Suchdielectric manipulations provide a force that may be used in a varietyof configurations or processes for the purpose of partitioning effectorcells and readout particles.

In yet another embodiment, acoustic waves, such as acoustic standingwaves, are used to trap, position and/or retain effector cells and/orreadout particles.

In yet another embodiment, effector cells and/or readout particles arestochastically loaded into the effector and readout zones of amicrofluidic chamber with high efficiency by appropriate design of thechamber geometry and the loading density. For example, FIG. 33 shows amicrofluidic chamber having an array of 45 microwells that arefabricated at the bottom of the chamber having a volume of approximately2 nanoliters. The loading of effector cells and readout particles intothe microwells of the microfluidic chamber, in one embodiment, isachieved using gravity as described above. If the number of effectorcells and readout particles is maintained sufficiently low there is alow probability that two effector cells and/or readout particles arepositioned within the same microwell within the microfluidic chamber.

The number of effector cells and readout particles per chamber can beselected such that the probability of having a chamber without both areadout particle and an effector cell is very low. As an example, in oneembodiment, the concentrations of effector cells and readout particlesare chosen such that there is an average of three of each type perchamber. Assuming a random distribution of particles within themicrowells the chance of having k particles of either type in a givenmicrowell of a chamber is given by Poisson statistics as:

P(more than one in a microwell)=λ^(k) e ^(−λ) /k!

where λ=the average number of particles per micro-well of themicrofluidic chamber, which in this example is (3+3)/45=0.133. Thus, theprobability of having 0 or 1 particles in a microwell in this embodimentis:

P(k=0)=e ^(−0.133)=87.5%, and P(k=1)=11.7%.

Thus, in this embodiment, the chance of having more than one particle isgiven by P(k>1)=100%−87.5%−11.7%=0.83%. The chance of having zeromicro-wells within a given chamber containing more than one particle maythen be calculated according to binomial statistics as:

P(no wells with more than one particle)=(1−0.0083)⁴⁵=69%.

Thus, in this example approximately 70% of the chambers will have nomicrowell with more than one particle. It will be appreciated that thisanalysis represents a lower bound to the fraction of useful chamberssince in chambers where some micro-wells have more that one particle inat least one micro-well a useful measurement will still be possibleusing the other chambers.

It is further considered that the micro-wells may be designed with asize such that they will not accommodate more than one particle. In thiscase the tipping of the device after loading may be used to ensure allparticles are contained within a micro-well of the device and that nomicro-wells have more than one particle. It is also understood thatdifferent types of particles, including different effector or readoutparticles, may be loaded in a sequential fashion or together dependingon the design of the assay. Finally, it is understood that thisgeometry, while using microwells, maintains the advantages of chamberisolation since the array of micro-wells is contained within a smallchamber that may have the capability for being isolated from otherchambers.

As noted above, the one or more of the methods described above, in oneembodiment, are combined are used to achieve segregation of a populationof cells optionally comprising one or more effector cells from apopulation of readout particles, in an effector zone and readout zone ofa microfluidic chamber, respectively. Additionally, one or more of themethods described above, in one embodiment, are used to achievesegregation of a cell subpopulation from an original cell population, orto segregate a subpopulation of readout particles from a population ofreadout zones, for example, in specific regions of an effector zone orreadout zone of a microfluidic chamber.

Effector cells and readout particles, in one embodiment, are deliveredto a chamber using a common inlet and segregated upon entry into thechamber. Alternatively, effector cells and readout particles areintroduced to the chamber via separate inlets specific for the effectorzone and readout zone. Referring to FIG. 47, a chamber 298 according toan embodiment of the invention is shown. Effector cells 290 areintroduced into effector zone 291 via effector cell inlet 292, whilereadout particles 293 are introduced into readout zone 294 of thechamber via readout particle inlet 295. In the illustrated embodiment,effector zone 291 and readout zone 294 have respective outlets 296 and297, however the invention is not limited thereto. Specifically, theeffector zone 291 and readout zone 294, in another embodiment, have acommon outlet.

In another embodiment, a compound chamber (i.e., a chamber comprising aplurality of subchambers) is used to provide an effector zone andreadout zone. One embodiment of a compound chamber 300 is shown at FIG.48. In one embodiment, a cell population comprising one or more effectorcells 301 is delivered to effector zone 302 defined by effector cellsubchamber 303 of compound chamber 300 via the cell inlet 304.Similarly, readout particles 305 are delivered to readout zone 306defined by readout particle subchamber 307 of compound chamber 300 viareadout particle inlet 308. In the illustrated embodiment (FIG. 48),effector cell subchamber 303 and readout particle subchamber 307 haverespective outlets 309 and 310. Effector cell subchamber 303 and readoutparticle subchamber 307 are in fluid communication via aperture 311.Valve 312, in one embodiment, is provided in aperture 311 to render theaperture reversibly sealable.

In one embodiment, adherence is used to separate effector cells fromreadout particles in some embodiments. For example, the skilled artisanis directed to the embodiment shown at FIG. 49. In FIG. 49, chamber 480is functionalized with a coating solution to enable adhesion ofanchorage-dependent readout cells. The chamber is then inverted to loadanchorage-dependent readout cells 481 until they adhere to top surface482. The chamber 480 is then inverted again to load a suspension ofcells 483, which are allowed to settle by gravity to bottom surface 484of the chamber 480, effectively separating the cell population and theanchorage-dependent readout cells 481 into separate effector and readoutzones. The cell population, in one embodiment, comprises one or moreeffector cells.

In the embodiment depicted in FIG. 50, a coating solution 530 promotingcell adhesion is introduced into half of chamber 531 by flowing thecoating solution and a second solution 532, e.g., phosphate bufferedsaline, in T junction 533 with laminar flow. Adherent readout ells 534are then introduced into chamber 531, and directed to the side withadherent coating 535 by gravity (represented by arrow). After anattachment period, unbound readout cells 534 are washed from the chamber531. A cell population 536 is then loaded in the chamber 531. The cellpopulation 536 is maintained on the opposite side 537 of the chamber 531by gravity while the adherent readout cells 534 stay on the side withadherent coating 535. The cell population, in one embodiment, comprisesone or more effector cells.

Referring to the embodiment depicted in FIG. 51, two solutions, e.g.,solution A 540 comprising an antibody against target A (anti-A antibody)and solution B 541 comprising an antibody against target B (anti-Bantibody), are loaded into chamber 542 from different sides of Tjunction 543, and allowed to coat the functionalized chamber 542 surface(e.g., protein-A coated PDMS). Using laminar flow, the first half 544 ofthe chamber 542 becomes coated with the anti-A antibody while the secondhalf 545 is coated with the anti-B antibody. Two different types ofparticles, first particle 546 displaying antigen A on its surface andsecond particle 547 displaying antigen B on its surface are thenintroduced into chamber 542. Segregation of the two types of particlescan be achieved by tilting chamber 542 first on one side, and then theother, so that once a particle reaches the section coated with theantibody against its displayed antigen, it remains on the proper side ofthe chamber.

Once a chamber is loaded with a cell population and a readout particleor readout particle population, and optionally additional reagent(s) forcarrying out an assay on the cell population, the chamber, in oneembodiment, is fluidically isolated from one or more remaining chambersof the microfluidic device. In one embodiment, isolation of the chamberis achieved by physically sealing it, e.g., using one or more valves tofluidically isolate the chamber from its surrounding environment. Aswill be understood by one of skill in the art, a valve as describedherein is controlled via a “control channel,” and by applying sufficientpressure to the control channel, a particular valve can be actuated. Inone embodiment, subsections (e.g., an effector zone and a readout zone)of a given chamber, or one subchamber of a compound chamber (i.e., achamber comprising a plurality of subchambers or wells), are isolatedfrom one another by physically sealing the subsections or subchambers,to fluidically isolate the subsections and/or subchambers, e.g., byusing one or more valves.

In another embodiment, isolation of a chamber from its surroundingenvironment is achieved without physically sealing the chamber. Rather,isolation is achieved by limiting the fluid communication betweenchambers to preclude significant contamination between one chamber andanother chamber of the microfluidic device. For example, instead ofusing a one or more valves, adjacent chambers are separated by the useof an immiscible fluid phase, such as an oil, to block chamber inletsand/or outlets. Alternatively, chambers are designed with inlets andoutlets such that the diffusion of molecules in and out of chambers issufficiently slow that it does not significantly impede the analysis ofeffect of secreted products within particular chambers.

Various chamber arrangements are described throughout.

FIG. 52 shows a chamber architecture for use with embodiments of theinvention. The architecture includes chambers 311 (fabricated in the“flow layer”) arranged in a column with each chamber isolated from itsneighbor by a valve 312 (“control channel” layer). In anotherembodiment, valves are located above each chamber (a “lid chamber”) asillustrated in FIG. 53, resulting in a higher on-chip feature densitythan the column architecture. In the embodiment depicted on the left ofFIG. 53, the width of the chambers 321 are less than the width of therounded feed channel 323 (e.g., to deliver effector cells and/or readoutparticles), in which case the valve 322 seals the perimeter of thechambers. Rounded channels, as discussed in detail herein, arefabricated with by molding PDMS on certain types of photoresist, such asMegaposit SPR220 Series (Microchem) and AZ 40 XT (MicroChemicals). Inthe embodiment depicted on the right of FIG. 53, the chamber 321 widthexceeds the width of the feed channel. In this embodiment, chambers areisolated by a single valve 322 that simultaneously closes the inlet andoutlet of each chamber.

Importantly, the microfluidic devices of the invention are not limitedto serially arranged chambers addressed in a “flow-through” mode wherechambers are arranged in columns in which the outlet of one chamberconnects to the inlet of an adjacent downstream chamber. Rather, anumber of different chamber arrangements and filling modes are providedherein and encompassed by the invention. For example, in one embodiment,chambers are arranged in parallel such that multiple chambers areaddressed simultaneously through the same feed channel. In a furtherembodiment, parallel chamber arrays are used in “dead-end” filling mode(i.e., where the chambers do not comprise an outlet).

FIGS. 54 and 55 show various parallel chamber arrangement embodiments ofthe invention. In these embodiments, each column of chambers 331 (FIG.54) and chambers 331 (FIG. 55) share a common inlet channel 332 andoutlet bus channel 333. In these configurations, cross-contaminationbetween chambers is prevented using valves 334. FIG. 55 shows anembodiment where an effector zone 335 of a chamber can be fluidicallyisolated from the readout zone 336 of the same chamber 331.Specifically, as illustrated in FIG. 55, the chamber 331, in oneembodiment, comprises a compound chamber in which an effector zone 335is separated from a readout zone 336 by sealable channel 337 (e.g.,sealable with a valve 337). Contamination between individual chambers,in one embodiment, is reduced as secreted products from each chamber arenot transported through chambers located further downstream on thedevice. Importantly, reagents in the feed channels can be replaced whilechambers remain isolated, eliminating the risk of gradient effects thatcould occur in serially arranged chambers.

In some embodiments, for example, as illustrated in FIG. 56, a singleconnection between a chamber and a channel, which may be controlled by avalve, can function as both an inlet and an outlet depending on thedirection of the flow, into or out of the chamber. Referring to FIG. 56,a chamber 341 is connected to a feed channel 342 via a single connectingchannel 343 that is under the control of a valve 344. In this case thechamber walls are made from an elastic and gas permeable material. Thisallows for the chamber to be “dead-end” filled through the connectingchannel 343 by pushing the air in the chamber 341 into the PDMSmaterial. A top-down view of this architecture is provided in the bottomleft of FIG. 56, where the chamber 341 is connected to the feed channel342 through a single connecting channel 343. A valve 344 can be actuatedto fluidically isolate the chamber 341 from the feed channel 342. Thetop left drawing in FIG. 56 shows a cross section of the samearchitecture.

Once the chamber 341 is filled, flow can still be directed into (FIG.56, top right) or out of (FIG. 56, bottom right) the chamber 341 bymodulating the pressure applied to the chamber, which causes the chamber341 to expand or compress in volume. The ability to modulate and changethe chamber volume is advantageous in the assaying of particles 345(e.g., cells) within the chamber 341. For example, in one embodiment,particles 345 are introduced into the chamber 341 by bringing them intothe feed channel 342, by applying a pressure to the feed channel 342that is higher than that of the chamber 341, and then opening the valve344 to the chamber 341 to allow the flow to enter the chamber 341. Inanother embodiment, the valve 344 is first opened and then the pressureof the feed channel 342 increased. Once inside the chamber 341, theparticles 345, e.g., effector cells or readout particles, will fall tothe bottom of the chamber under the effect of gravity.

In a further embodiment, pressure of the feed channel 342 is thenreduced, causing the chamber 341 to relax back to its original volumewith the flow to be directed out of the chamber. Since the particles 345(e.g., effector cells and/or readout particles), are at the bottom ofthe chamber 341, they are substantially removed from the flow and remainin the chamber when the pressure is reduced. Feed channel 342 pressuremodulations may also be used to periodically add fresh medium to thechamber 341 in order to maintain the viability and growth of cells,whether effector cells or readout cells. In this embodiment, the chamber341 is inflated with fresh medium which mixes with the medium already inthe chamber by diffusion. Once mixed, a portion of the fluid inside thechamber 341 is removed and the process repeated as desired by the user.Importantly, by flushing the feed channel 342 with the chamber 341closed to the feed channel between subsequent steps of medium addition(e.g., by actuation of valve 344), the contents of a chamber within anarray do not contaminate other chambers in the chamber array.

This same approach of feed channel pressure modulations, in oneembodiment, is used to add reagents to a chamber that are required orsufficient to observe and measure the effect of one or more effectorcells on one or more readout particles within the chamber. For instance,and described in detail below, a cell population comprising one or moreeffector cells is assayed in one embodiment, for the ability of the oneor more of the effector cells to neutralize a cytokine. In this case, achamber may be first loaded with the cell population, for example a cellpopulation comprising one or more effector cells that secreteantibodies. The antibodies are tested within the chamber for theirability to neutralize a cytokine. For example, in one embodiment, thecytokine neutralization effect is measured by providing readoutparticles to the chamber, whereby the readout particles comprise cellsthat are responsive to the cytokine, for instance by expression of afluorescent protein. In one embodiment, individual chambers are firstisolated and incubated to allow for the accumulation of a sufficientamount of the antibody. A volume of medium containing the cytokine isthen added to the chambers by inflating them, thereby maintaining theantibodies in the chambers and not allowing for cross-contaminationbetween chambers. The chambers, in a further embodiment, are thenincubated with additional volume exchanges as required to determine ifthe chamber contains an effector cell that secretes an antibody that iscapable of neutralizing the cytokine.

The above strategy may also be achieved without the use of mechanisms tomodulate the volume of chambers. For example, a chamber, in oneembodiment, is constructed to have two separate compartments (e.g.,separate chambers, or subchambers or wells within an individual chamber)that are independently flushed with reagents, and which may also beisolated from other chambers on the device. The exchange of medium withfresh medium, or medium containing other components/reagents needed fora particular assay, in one embodiment, is implemented by isolating onecompartment of a chamber, e.g., a “reagent compartment,” from the othercompartment, where the “other compartment” contains the effector cellsand readout particles, flushing the reagent compartment, and thenreconnecting the compartments to allow for mixing by diffusion or byanother means such as pumping between the two compartments.

As discussed herein, an effector zone and readout zone of a chamber, inone embodiment, are fluidically isolated from one another via one ormore valves. This architecture can be further extended to chambers thatare dead-end filled and in communication with each other. As illustratedin FIG. 57, separating the effector zone 351 and the read-out zone 352,e.g., by valves 353, offers the advantage of individual addressability:reagents in each zone may be exchanged independently and/or assays maybe subsequently performed on both the effector cells and read outparticles. Note that the dead end portion of the channel is not depictedin FIG. 57.

As provided throughout, in one aspect, the present invention relates toa method of identifying a cell population comprising an effector cellhaving an extracellular effect and in another aspect, methods areprovided for identifying a cell population having a variation in anextracellular effect. Once it is determined that the cell populationdemonstrates the extracellular effect, or a variation of anextracellular effect, the cell population or portion thereof isrecovered to obtain a recovered cell population. Recovery, in oneembodiment, comprises piercing the microfluidic chamber comprising thecell population comprising the one or more cells that exhibit theextracellular effect, with a microcapillary and aspirating the chamber'scontents or a portion thereof to obtain a recovered aspirated cellpopulation.

The recovered cell population(s), once recovered, in one embodiment, aresubjected to further analysis, for example to identify a single effectorcell or a subpopulation of effector cells from the recovered cellpopulation that is responsible for the variation in the extracellulareffect. The recovered cell population(s) can be analyzed in limitingdilution, as subpopulations for a second extracellular effect, which canbe the same or different from the first extracellular effect. Cellsubpopulations having the second extracellular effect can then berecovered for further analysis, for example for a third extracellulareffect on a microfluidic device, or by a benchtop method, for exampleRT-PCR and/or next generation sequencing.

Various methods for the recovery of one or more cells from a specificchamber(s) are amenable for use herein.

The PDMS membrane design of the devices provided herein enables theselective recovery of cells from any chamber by piercing the uppermembrane with a microcapillary. In one embodiment, cell recovery from achamber is carried out based in part on the methods set forth by Lecaultet al. (2011). Nature Methods 8, pp. 581-586, incorporated by referenceherein in its entirety for all purposes. The membrane above a particularchamber is pierced with the microcapillary and cells are aspirated (FIG.58, top). The same microcapillary can be used to recover multiple cellpopulations on one device. Recovered cells can then be deposited inmicrofuge tubes for further analysis, for example, RT-PCR analysis orsubjected to a further functional assay on the same microfluidic deviceor a different microfluidic device.

In one embodiment, once effector cells from identified chambers arerecovered, they are reintroduced into the same device at a differentregion, at limiting dilution (e.g., either a single cell per chamber orsmaller populations than the initial assay) to determine which effectorcell(s) is responsible for the variation in the extracellular effect,i.e., by performing another extracellular assay on the recovered cells(see, e.g., FIG. 2).

In one embodiment, one or more cell population are recovered with amicrocapillary by aspirating the contents of the chamber containing thecell population to provide a recovered aspirated cell population. In afurther embodiment, recovered aspirated cell population is reinjectedinto a microfluidic device with the microcapillary, wherein themicrocapillary pierces one wall of the microfluidic device. Pressure isthen applied to the microcapillary to flow the recovered aspirated cellpopulation into separate chambers of the microfluidic device, and themicrocapillary is retracted to cause the wall of the microfluidicstructure to substantially re-seal.

Recovery, in one embodiment is automated and using a roboticmicrocapillary instrument (FIG. 58, bottom). However, recovery can alsobe accomplished manually with a microcapillary. The recovery methodsprovided herein allow for the recovery from 100 chambers with >95%efficiency in 15 minutes. Alternatively, the recovered effector cellscan be introduced into a second device or analyzed via benchtop methodsto determine the identity of particular cell(s) responsible for thevariation in the extracellular effect.

A microcapillary, as stated above in one embodiment, is used to recoverone or more cell populations (or subpopulations, depending on whether amicrofluidic enrichment has taken place) from a microfluidic chamber.and aspirating the chamber's contents or a portion thereof to obtain arecovered aspirated cell population. The cells in the one or more cellpopulations (or subpopulations) are substantially recovered byaspirating the chamber contents into the microcapillary, to provide arecovered aspirated cell population (or subpopulation). Themicrocapillary in one embodiment, has a diameter of from about 5 μm toabout 200 μm. In a further embodiment, the microcapillary has a diameterof from about 5 μm to about 200 μm, or from about 5 μm to about 150 μm,or from about 5 μm to about 100 μm, or from about 5 μm to about 75 μm,or from about 5 μm to about 50 μm, or from about 50 μm to about 200 μm,or from about 100 μm to about 200 μm, or from about 150 μm to about 200μm.

In some embodiments, the microcapillary has a beveled tip. In someembodiments, the microcapillary has an oval, square or circular crosssection. Additionally, as shown in FIG. 58, the microcapillary in someembodiments is mounted on a robotic micromanipulation system on amicroscope to provide an automated recovery apparatus.

In one embodiment, the microcapillary provided herein has a singlebarrel. However, the microcappilary in other embodiments has multiplebarrels, for example a double barrel, a triple barrel, or more thanthree barrels.

A cell or cells can also be selectively recovered by using microfluidicvalves to uniquely address the specified chamber and to direct flow toflush the single cell or multiple cells from the chamber to an outletport for recovery. Cell adherence to the device substrate, in oneembodiment, can be minimized by methods known to those of skill in theart, for example, purging or coating the microfluidic substrate withTrypsin. In another embodiment, a plurality of chambers comprising cellsof interest are simultaneously recovered through a single port, e.g.,via the use of addressable valve arrays to control fluid flow. Inanother embodiment, cells from chambers that are not of interest arefirst removed from the device, either by washing or lysis. The remainingcontents of the device including the cells of interest are thenrecovered by flushing to a desired port.

In one embodiment, the contents of a chamber comprising an effector celldisplaying a variation in an extracellular effect are recovered from thedevice by aspiration, for example, by using a microcapillary fabricatedto have an appropriate size and shape. In some embodiments, the recoverymethod comprises piercing the top of the chamber comprising the cell(s)of interest with the microcapillary and aspirating the cell(s) ofinterest. In one embodiment, the membrane reseals or substantiallyreseals after piercing is complete. In another embodiment, recovery ofthe contents of a chamber comprising an effector cell displaying avariation in an extracellular effect (e.g., one or more ASCs) isperformed by first cutting a wall of the chamber to create an accesspoint and then extracting cells by aspiration using a microcapillary. Inyet another embodiment, the microfluidic device used to assay theextracellular effect is fabricated such that the chambers are exposed bypeeling away the material on one wall, thereby leaving an openmicro-well array. Identified chambers (i.e., chamber(s) comprising aneffector cell displaying a variation in an extracellular effect) arethen aspirated from their respective chambers. In order to facilitatethe precise extraction of microfluidic well contents, aspiration toolssuch as microcapillary tubes, in one embodiment, are mounted on arobotic micromanipulator, or a manual micromanipulator (FIG. 58).However, aspiration in other embodiments is performed manually.

In some cases, it is desirable to extract a subset of cells from a givenchamber. For instance, methods provided herein allow for the removal ofcells from a specific region of a microfluidic chamber, for example, areadout zone or an effector zone. In one embodiment, the recovery methodprovided herein comprises aspiration of individual single cells in aserial manner.

Recovery of one or more cells from one or more microfluidic chambers, inone embodiment, comprises magnetic isolation/recovery. For example, inone embodiment, a microfluidic chamber is exposed to a magnetic particle(or plurality of magnetic particles) that adheres to the one or morecells within the chamber. Adherence can be either selective for a singlecell, a subpopulation of the population of cells in the well(s), ornon-selective, i.e., the magnet can adhere to all cells. In this case,instead of aspirating cells into a microcapillary, cells labeled withmagnetic particles are drawn to a magnetic probe that creates a magneticfield gradient. The probe, in one embodiment, is designed to enable themagnetic field to be turned on and off, causing cells to adhere to itfor removal and then be released during deposition. (EasySep SelectionKit, StemCell Technologies).

Single cells or a plurality of cells harvested from chambers, in oneembodiment, are deposited into one or more receptacles for furtheranalysis, for example, open micro-wells, micro-droplets, tubes, culturedishes, plates, petri dishes, enzyme-linked immunosorbent spot (ELISPOT)plates, a second microfluidic device, the same microfluidic device (in adifferent region), etc. The choice of receptacle is determined by one ofskill in the art, and is based on the nature of the downstream analysisand/or storage.

In some embodiments, cell-derived products or intracellular materialsare recovered from microfluidic chambers of interest, alternatively orin addition to the recovery of a single cell or plurality cells. Forexample, if a microfluidic chamber is identified as having a cell thatdemonstrates a variation in an extracellular effect, in one embodiment,the secretion products from the chamber are is recovered for downstreamanalysis (e.g., sequence analysis). In another embodiment, the cell orplurality of cells is lysed on the microfluidic device, e.g., within thechamber that the first assay is performed, and the lysate is recovered.In one embodiment, the lysate is subjected to further on chipprocessing, for example, to isolate protein, mRNA or DNA from the cellor plurality of cells. The RNA of the cell or plurality of cells withina single chamber, in one embodiment, is selectively recovered by usingmicrofluidic valves to flush through a specified chamber using a reagentthat causes the release of the RNA from the cells. This material is thencollected at the outlet port. In another embodiment, the cells in allwells or a subset of wells are lysed using a lysis reagent, and then thecontents of a given chamber or subset of chambers are recovered. Inanother embodiment, the cells within a chamber of interest or chambersof interest are lysed in the presence of beads that capture the RNAreleased from the cells followed by recovery of the beads, for example,by using the techniques described above for cell recovery. In this casethe RNA may also be converted to cDNA using a reverse transcriptaseenzyme prior to or subsequent recovery. One example of how to accomplishon chip mRNA isolation, cDNA synthesis and recovery can be found inAnal. Chem 78 (2006), pp. 3084-3089, the contents of which areincorporated by reference in their entirety for all purposes.

Following the recovery of cells or cell-derived materials from a chamberor chambers of interest, these materials or cells are analyzed toidentify or characterize the isolate or the single cell or plurality ofcells. As mentioned above, further analysis can be via a microfluidicassay (see, e.g., FIG. 2), or a benchtop assay. The present inventionallows for multiple rounds of microfluidic analysis, for example toidentify a cell subpopulation from a recovered cell population thatdisplays a second extracellular effect, a third extracellular effect andor a fourth extracellular effect. By repeating the extracellular effectassays on recovered cell populations, the user of the method obtainshighly enriched cell populations for a functional feature of interest,or multiple functional features of interest.

In one embodiment, one or more cell populations exhibiting theextracellular effect or variation in the extracellular effect arerecovered to obtain one or more recovered cell populations. Once one ormore individual cell populations are identified and recovered, the oneor more individual cell populations are further analyzed to determinethe cell or cells responsible for the observed extracellular effect. Inone embodiment, the method comprises retaining a plurality of cellsubpopulations originating from the one or more recovered cellpopulations in separate chambers of a microfluidic device. Each of theseparate chambers comprises a readout particle population comprising oneor more readout particles. The individual cell subpopulations areincubated with the readout particle population within the chambers. Theindividual cell subpopulations are assayed for a variation of a secondextracellular effect, wherein the readout particle population orsubpopulation thereof provides a readout of the second extracellulareffect. The second extracellular effect is the same extracellular effector a different extracellular effect as the extracellular effect measuredon the recovered cell population. Based on the second extracellulareffect assay, one or more individual cell subpopulations are identifiedthat exhibit a variation in the second extracellular effect. The one ormore individual cell subpopulations in one embodiment, are thenrecovered for further analysis. The second extracellular effect assay isone of the extracellular effect assays described herein.

One or more individual cell subpopulations are recovered, for example,with a microcapillary, as described in detail above. The microcapillary,in one embodiment, is used to reinject recovered cell subpopulationsinto the same microfluidic device, or a different microfluidic device,to further enrich for a population of cells displaying an extracellulareffect. For example, a plurality of cell subpopulations originating fromthe recovered cell subpopulation, in one embodiment, are retained inseparate chambers of a microfluidic device, wherein each of the separatechambers comprises a readout particle population comprising one or morereadout particles. The cell subpopulations are incubated with thereadout particle populations within the microfluidic chambers and thecell subpopulations are assayed for the presence of a thirdextracellular effect. The readout particle population or subpopulationthereof provides a readout of the third extracellular effect. Based onthe results of the assaying step, it is determined whether one or morecells within one or more of the cell subpopulations exhibits the thirdextracellular effect. The one or more cell subpopulations can then berecovered as described herein.

In one embodiment, cells from a recovered cell population or recoveredcell subpopulation or plurality of cell populations or subpopulationsare retained in a plurality of vessels as cell subpopulations. The termcell sub-subpopulation is meant to refer to a subpopulation of analready recovered cell subpopulation. However, one of skill in the artwill recognize that a cell subpopulation can be partitioned into furthersubpopulations, and the use of the term “sub-subpopulation” is notnecessary to make this distinction. Each cell subpopulation is presentin an individual vessel. The individual subpopulations orsub-subpopulations are lysed to provide and one or more nucleic acidswithin each lysed cell subpopulation or lysed cell sub-subpopulation areamplified. In a further embodiment, the one or more nucleic acidscomprise an antibody gene.

Several approaches including microfluidic analysis may be used for thisdownstream analysis, depending on the nature of the cells, the number ofcells in the original screen, and the intent of the analysis. In oneembodiment, where a population effector cells is recovered from achamber, or a plurality of populations are recovered from multiplechambers, each cell of the plurality is isolated into an individualvessel (e.g. individual microfluidic chamber) and analysis is performedon each effector cell individually. The individual cell analysis can bea microfluidic analysis (FIG. 2), or a benchtop analysis. In anotherembodiment, where a population effector cells is recovered from achamber, or a plurality of populations are recovered from multiplechambers, the cell populations are reintroduced onto the samemicrofluidic device in a separate region (or a second device), and thecells are isolated at a limiting dilution, i.e., as “cellsubpopulations,” for example, the cells are isolated at a density of asingle cell per chamber, or from about two to about ten cells perchamber and a second extracellular effect assay is performed. Thedownstream analysis (microfluidic or otherwise) may be on any size cellsubpopulations, for example, the same size as the initial extracellulareffect cell assay, or a smaller population size, e.g., a single cell,two cells, from about two cells to about 20 cells, from about two cellsto about 25 cells. Readout particles are introduced into the chamberscomprising the cell subpopulations, and the second extracellular effectassay is performed. The contents of chambers that comprise a cellsubpopulation displaying a variation in the extracellular effect areharvested for further analysis. This further analysis can bemicrofluidic analysis (e.g., by performing a third extracellular effectassay, single cell PCR), or a benchtop analysis (e.g., PCR, nextgeneration sequencing).

In one embodiment, individual recovered effector cells are expanded inculture by distributing the plurality of cells at limiting dilution intoa plurality of cell culture chambers in order to obtain clones from therecovered cells. For example, in an embodiment where a plurality ofeffector cells of a cell line engineered to express a library ofantibodies are present in a chamber or chambers of interest, the cellsfrom the chamber or chambers are subjected to limiting dilution in orderto isolate single effector cells that were present in the chamber. Thesingle effector cells are then used to obtain clonal populations of eachrespective effector cell. One or more of the clonal populations can thenbe analyzed to asses which effector cell produces the antibody ofinterest by measuring the properties of the antibodies secreted (e.g.,by ELISA or a functional assay).

Alternatively or additionally, cells are recovered from a microfluidicchamber, isolated, e.g., by limiting dilution, and expanded to obtainsufficient material for the sequencing or amplification and purificationof one or more genes of interest, e.g., a gene that encodes an antibodyof interest. In yet another embodiment, cells are recovered from amicrofluidic chamber, isolated, e.g., by limiting dilution, and used forsingle-cell DNA or mRNA amplification, e.g., by the polymerase chainreaction (PCR) or reverse transcriptase (RT)-PCR, followed bysequencing, to determine the sequence of one or more genes of interest.In even another embodiment, cells of interest are recovered from one ormore microfluidic chambers, isolated, e.g., by limiting dilution, andsubsequently used for single-cell DNA or mRNA amplification of the genesof interest, followed by the cloning of these genes into another celltype for subsequent expression and analysis.

In one embodiment, the recovered cell population(s) or subpopulation(s)may be isolated and used for an in vivo analysis, for example byinjecting them into an animal, or exapanded in culture followed bysubjecting the cells to one or more functional assays previously carriedout on the microfluidic device.

In one embodiment, a cell population or subpopulation is recovered thatdisplays a variation in an extracellular effect (e.g., after a first orsecond microfluidic extracellular effect assay) and one or more nucleicacids of the cells are subjected to amplification. Amplification, in oneembodiment, is carried out by the polymerase chain reaction (PCR),5′-Rapid amplification of cDNA ends (RACE), in vitro transcription orwhole transcriptome amplification (WTA). In a further embodiment,amplification is carried out by reverse transcription (RT)-PCR. RT-PCRcan be on single cells, or a plurality of cells of the population.Although single cell RT-PCR is becoming common place as an analyticalmethod, the amplification of antibody genes presents a nontrivialchallenge due to multiple gene usage and variability. Two mainapproaches for recovery of antibody genes from single cells includeRT-PCR using degenerate primers and 5′ rapid amplification of cDNA ends(RACE) PCR. In one embodiment, the RT-PCR method is based ongene-specific template-switching RT, followed by semi-nested PCR andnext-generation amplicon sequencing.

One embodiment of an RT-PCR method for use with identified effectorcells having a variation in an extracellular effect is shown at FIG. 59.The schematic shows a single cell HV/LV approach using templateswitching reverse transcription and multiplexed primers. In thisembodiment, single cells are deposited into microfuge tubes and cDNA isgenerated from multiplexed gene-specific primers targeting the constantregion of heavy and light chains. Template-switching activity of MMLVenzyme is used to append the reverse complement of a template-switchingoligo onto the 3′ end of the resulting cDNA (Huber et al. 1989). J.Biol. Chem. 264, pp. 4669-4678; Luo and Taylor (1990). J. Virology 64,pp. 4321-4328; each incorporated by reference in their entireties forall purposes). Semi-nested PCR (common 3′ primers and multiplexed nestedprimers positioned inside the RT primer region) using multiplexedprimers at constant region of heavy and light chain, and a universalprimer complementary to the copied template switching oligo, is used toamplify cDNA and introduce indexing sequences that are specific to eachsingle cell amplicon. The resulting single cell amplicons are pooled andsequenced.

In some cases, a recovered cell population, following recovery from themicrofluidic device, is not further isolated into single cells. Forexample, if the plurality of cells isolated from a chamber contain acell that secretes an antibody of interest (e.g., present in apopulation of one or more additional cells that secrete otherantibody(ies)), the plurality of cells, in one embodiment, is expandedin culture to generate clonal populations of the cells of the plurality,some of which make the desired product (i.e., the antibody of interest).In another embodiment, a plurality of cells isolated from a microfluidicwell is lysed (either on the microfluidic device or subsequent torecovery), followed by amplification of the pooled nucleic acidpopulation and analysis by sequencing. In this case, a bioinformaticsanalysis of the sequences obtained may be used to infer, possibly usinginformation from other sources, which of the sequences is likely toencode for the protein of interest (for example, an antibody).Importantly, the analysis method afforded by the present invention isgreatly simplified, as compared to bulk analysis of a large numbers ofcells, due to the limited number of cells that are recovered. Thislimited number of cells provides a reduced complexity of the genomicinformation within the population of cells.

In one embodiment, the amplified DNA sequences of the plurality of cellsare used to create libraries of sequences that are recombinantlyexpressed in an immortalized cell line, according to methods known tothose of skill in the art. This cell line may then be analyzed, possiblyby first isolating clones, to identify the genes of interest. In someinstances these libraries are used to screen for combinations of genesthat result in a protein complex of interest. For example, in oneembodiment, genes expressed in cells of interest include both heavy andlight chains of antibody genes in order to identify gene pairings thathave the desired properties. The complexity of such analyses is greatlyreduced by the fact that the number of cells analyzed in the originalscreen is small. For example, if there are 10 cells in the originalscreen there are only 100 possible antibody heavy and light chainpairings. By comparison, bulk samples typically have thousands ofdifferent antibody sequences, corresponding to millions of possiblepairings.

In some instances, recovered cells may contain different cell types thatcan be isolated using methods known to those of skill in the art. Forinstance, if the microfluidic chamber comprises both antibody secretingcells and fibroblasts, used to maintain the antibody secreting cells,the antibody secreting cells in one embodiment, are separated from thefibroblasts after recovery using affinity capture methods.

EXAMPLES

The present invention is further illustrated by reference to thefollowing Examples. However, it should be noted that these Examples,like the embodiments described above, are illustrative and are not to beconstrued as restricting the scope of the invention in any way.

Example 1—Fabrication of Microfluidic Devices

Microfluidic devices were fabricated using the protocol described inLecault et al. (2011). Nature Methods 8, pp. 581-586 or an adaptedversion of this protocol (e.g., modified baking times or curingprotocols) to obtain multiple layers including, from the bottom glassslide to the top: a flow chambers, a control layer, a membrane, and bathlayer (FIG. 27). A cross-section of a device and a corresponding3D-schematic of the chambers, flow channels and control channels areshown in FIG. 61 and FIG. 62, respectively. As described herein, where acontrol channel crosses a flow channel, a pinch valve is formed, andclosed by pressurizing the control channel. A cover layer was added incertain instances to close the bath, or left omitted for an open bathsuch as the one shown in the device on FIG. 63.

Example 2—Microfluidic Device for Cell Enrichment by Selection andReinjection

The microfluidic device shown in FIG. 63 was used to implement aneffector cell (antibody secreting cell) selection assay with reinjectioncapabilities for enrichment. As shown in FIG. 64, the device includessix inlet ports 1030, six control ports 1031 for controlling each inlet,four reinjection ports, one of which is 1032 and a single output port1033. The device contains an array of 8192 identical unit cells, one ofwhich is depicted in FIG. 2B and FIG. 61. Each unit cell contains achamber which is 100 μm in width, 160 μm in length and 160 μm in height.Each chamber has a volume of about 2.6 nL. The device is divided intofour sub-arrays, one of which is indicated by 1032 (FIG. 64). Eachsub-array contains 2048 of the 8192 total unit cells. Each sub-array hasits own sub-array valve 1034, which allows each sub-array to befluidically addressed independently of the other sub-arrays. Eachsub-array has a single reinjection port 1035. The reinjection ports arecircular chambers having diameters of about 300 μm, depths of about 160μm and volumes of about 11.3 nL. The reinjection ports are pierced by amicrocapillary in order to inject reagents or particles into one or moreof the four sub-arrays.

Example 3—Microfluidic Device for Segregation of Effector and ReadoutCells

A microfluidic device was designed to permit readout cells to be kept inseparate, isolated chambers from effector cells and to allow thecontents of these chambers to be mixed on demand when needed. Thisconfiguration is used to implement effector cell assays, for example,cytokine neutralization assays. The architecture is particularly usefulto prevent an effector cell's secretion product (e.g., antibody) frombeing completely washed away when the chamber comprising the effectorcell(s) is perfused with a fluid, for example, when provided with freshculture medium to maintain viability over extended periods of time. Thisconfiguration is also amenable if it is useful to limit the time duringwhich the effector cell(s) is exposed to the medium conditions requiredfor the extracellular effect assay (e.g., when a toxic cytokine is usedas an accessory particle).

As shown in FIG. 65, this device consists of six inlet ports 1040, ninecontrol ports 1041 and 1042, two outlet ports 1043 and 1044, and anarray of 396 unit cells, each identical to 1045 and illustrated in moredetail in FIG. 66. Each unit cell contains two circular chambers. Eachchamber has a diameter of 210 μm, a height of 180 μm and a volume of 5.6nL. Typically, one of these chambers holds one or more effector cell andthe other chamber holds one or more readout cells. A flow channelconnects the two chambers. This flow channel can be closed with the“diffusion valve” 1050. With the diffusion valve 1050 open, the contentsof the two chambers mix via diffusion. With the diffusion valve 1050closed, the chambers are isolated from each other. Two flow channelsserially connect each unit cell to each other unit cell in the device.One of these flow channels flows through chamber 1 and the other flowsthrough chamber 2. These two flow channels are closed with “main valve1” 1051 and “main valve 2” 1052 to isolate the chambers of each unitcell from the chambers of each other unit cell. These two valves areopened and closed independently, permitting independent perfusion ofchamber 1 or chamber 2. Cross sections of the device in the vertical andhorizontal planes (dotted lines) from FIG. 66 are shown in FIG. 67 andFIG. 68, respectively.

An example of how a cytokine neutralization functional assay which canbe implemented with the device is shown in FIG. 69. Panel A shows thetop chamber containing at least one effector cell and the bottom chambercontaining a plurality of readout cells. Both chambers contain an equalconcentration of cytokine, required to maintain the viability of readoutcells. All valves are closed. Closed valves are illustrated shaded blackand opened valves are illustrated with horizontal hatched lines. Panel Bshows that time has passed and the at least one effector cell hassecreted antibodies. In the particular case that is illustrated, theantibodies bind to the cytokines in the effector chamber, neutralizingthem. Panel C shows that the diffusion valve 1050 has been opened. Freecytokines diffuse from the bottom chamber towards the top chamber.Neutralized cytokines diffuse from the top chamber towards the bottomchamber. Panel D shows that enough time has passed such that theconcentration of free and neutralized cytokines in each chamber hassubstantially equalized. All valves are then actuated and locked. Theeffective concentration of cytokine in the bottom chambers comprisingthe readout cells is now about half the initial concentration. Panel Eshows that effector cells have been perfused with fresh mediumcontaining additional cytokine. The perfusion has removed theneutralized cytokine from the top chamber. The process is repeated everyfew hours, eventually leading to a complete depletion of cytokine anddeath of readout cells. In other cases, the effect of cytokineneutralization on readout cells may include activation or inhibition ofa signaling pathway, growth arrest, differentiation, or a change inmorphology. The effect on the readout cells is then measured by methodsknown to those of skill in the art.

Example 4—Integration of Cell Fence Chambers

Devices having cell fences for capture of a single effector cell or aheterogeneous population of cells comprising one or more effector cellswere fabricated as follows.

PDMS devices with cell fences were fabricated by molding from a reusableflow channel mold and reusable control channel. The flow channel moldcomprised multiple layers of photoresist on a silicon wafer substrate.The bulk of the chambers in the devices utilized herein were defined byan SU-8 100 feature which sits directly on the Si wafer. Cell fences,when fabricated, were defined by a thinner SU-8 2010 feature which saton top of the SU-8 100 feature. This is depicted schematically in FIG.31.

The majority of microfluidic chambers were fabricated using SU-8 100negative photoresist (typically 160 μm in height) following standardprotocols, except that the final step, development, was omitted. Thewafers were cooled after the post-exposure bake, and then SU-8 2010 wasspun on top of the undeveloped SU-8 100, typically at a height 10-20 μm.The thickness of the SU-8 2010 determined the height of the fence. Thedepth of each chamber was the combined height of both photoresistlayers.

In alternative fabrication procedures, the SU-8 100 layer is fullydeveloped, and then the wafer is coated with an additional layer of SU-8100 which is higher than the first layer, the difference in heightbecoming the height of the fence.

The molds were then parylene coated (chemical vapor depositedpoly(p-xylylene) polymers barrier) to reduce sticking of PDMS duringmolding, enhance mold durability and enable replication of smallfeatures that could not be produced with the aforementioned parylenecoating.

A number of prototype devices were fabricated to determine feasiblefence width-height combinations. A prototype flow layer mold wasfabricated in order to determine dimensions which could successfully befabricated. This mold consisted of multiple chambers with fences, withthe width of the fences being varied across the same mold. Also, theheight of the fence and of the chamber could be varied duringfabrication (by adjusting spin speeds). The prototype molds containedonly chambers for fabrication testing.

Two photolithographic masks were required, one to define the bulk of thechambers (SU-8 100) and the other to define the fences (SU-8 2010).However, one physical mask could be used, containing both sets offeatures.

A prototype fence chamber was fabricated where the full width W of thechamber and was fixed at 300 μm. The typical width of chambers on mostexisting devices is 160 μm. Initially, each half of these chambers wasmade a similar size to allow a separate inlet and outlet for each of theeffector and readout zones. L is the length of the chamber. For eachunique value of X, there were three chambers with L=160 μm (length) andone more with L=2000 μm. The chamber with extended length was includedin order to allow the final PDMS mold to be easily cut perpendicular tothe fence, so that the cross-section could be imaged.

Initially a single mold was fabricated, providing data on a single setof chamber and fence heights. Table 5 summarizes the dimensions of theprototype mold:

TABLE 5 Prototype mold dimension summary W (design value) 300 μm L(design value) 160 / 2000 μm SU-8 100 Height (measured) 160 ± 5 μm SU-82010 Height (measured)  17 ± 1 μm

Table 6 lists the fence widths which were on the mold and which oneswere successfully fabricated:

TABLE 6 X (fence X (fence width) width) [μm] [μm] Successful DesignMeasured PDMS Value Values Fabrication? 5.0 can't measure No 7.5 can'tmeasure No 10 18 ± 2 No 12.5 21 ± 2 No 15 25 ± 2 No 17.5 29 ± 2 Yes 2031 ± 2 Yes 25 37 ± 2 Yes 30 42 ± 2 Yes 35 48 ± 2 Yes

The table above shows that the actual fence widths turned out largerthan the design values.

Fabrication failures were not obvious until after PDMS molding. For thesmaller fence widths, it was difficult to determine the quality of thephotoresist mold by viewing it under the microscope. Fences failedbecause the PDMS making up the fences either stayed in the moldcompletely or partially ripped away from the bottom of the chambers. Forexample, a fence having a width of 15 μm failed. The minimum width whichcould be fabricated using this particular design and the above describedmethods was 17.5 μm.

It was observed during PDMS molding of the prototype devices that thefence often tore apart from the chamber bottom. This tearing appeared tobegin at the 90 degree corner where the fence met the wall of thechamber. This 90 degree corner is a stress concentration. The design wasmodified to include 45 degree chambers in order to reduce these stressconcentrations and thereby improve PMDS molding capability. This designchange allowed the fence width to be reduced below the 17.5 μm width,which was the largest size possible on the testing mold. The width ofthe fence was reduced to a design value of 10 μm. The actual measuredvalue, 12 μm, was much closer to the design value than was achieved onthe prototype mold.

Fences in this example run parallel to the flow. However, there is noinherent reason that fences cannot be fabricated to be perpendicular tothe flow or diagonal relative to the flow. Similarly, chambers in thisexample are symmetric. However, the invention is not limited thereto.

Individual microfluidic chambers having a cell fence, an effector zoneand a readout zone were loaded with a cell population and readoutparticles using a tilting method to direct the effector cells to theeffector zone or the readout particles to the readout zone.

FIGS. 70A, 70C, and 70E show light microscopy images of microfluidicchambers having cell fences and FIGS. 70B, 70D, and 70F, show theequivalent chambers under fluorescence microscopy. The beads (readoutparticles) in the readout zone show an aggregation of effector cellproduct on the beads that is generally uniform despite relative distancefrom the effector cells and the effector zone.

Example 5—Robust Microfluidic Growth of Antibody-Secreting EffectorCells

A plurality of recombinant Chinese Hamster Ovary (CHO) cells (effectorcells) producing a human IgG antibody was loaded in the microfluidicdevice at a concentration of 2 M cells/mL. Secreted antibodies werecaptured on protein A-coated beads (average diameter: 4.9 μm) during a 2hour incubation followed by the addition of Dylight 594-conjugatedF(ab′)₂ fragment of rabbit anti-human IgG (H+L) and washing (FIG. 71).Bright field and fluorescent images were taken to quantify antibodysecretion and capture. Cells were subsequently cultured in the devicefor 4.5 days to generate clonal populations and clones secreting highamounts of antibody were recovered from the device for furtherexpansion.

FIG. 72 shows time-lapse imaging of a CHO clone after the antibodydetection effector cell assay was performed. The readout beads areidentified by the black arrow while the cells are identified by thewhite arrow. FIG. 73 shows growth curves (error bars, s.d.) of CHO cellscultivated in shake flasks (n=3 experiments in triplicate seeded at2.5×10⁵ cells m^(ml-1)) as single cells in 96 well plates (n=3experiments; 27-36 clones per plate) or in the microfluidic array (n=3experiments; 50 clones tracked per experiment). CHO cells expanded inthe microfluidic array exhibited growth rates that were comparable tobatch shake flask cultures and superior to single cells cultured in96-multiwell plates (FIG. 73).

Example 6—Sensitivity of Single ASC Detection in the Presence of aHeterogeneous Population of ASCs

Mathematical modeling was used to determine the total efficiency ofantibody capture on beads within a chamber having a volume of 4.1 nL(160 μM×160 μM×160 μM) as a function of time and as a function of thenumber of beads. The average secretion rate for this line of CHO cellswas measured on bulk samples to be approximately 300 proteins persecond. This secretion rate is comparable to the expected secretion ratefrom primary antibody secreting cells which are estimated to secretebetween 100 and 2000 antibodies per second. Thus, based on thesemeasurements it was determined that the beads furthest away from thecell are expected to capture approximately 4000 antibodies during a twohour incubation, and is detected via fluorescence.

In the example shown in FIG. 74A to 74D, the target of interest(antigen) is hen egg white lysozyme (HEL). A device was loaded witheffector cells derived from two hybridoma lines, HyHel5 and 4B2. HyHel5hybridoma cells secrete a monoclonal antibody that binds HEL and 4B2hybridoma cells secrete a monoclonal antibody that does not bind HEL.Referring to FIG. 74A, the top panel shows a HyHel5 cell loaded into achamber of a device having approximately 700 assay chambers. HyHel5 cellwere initially loaded at a density that results in an average ofapproximately 1 cell every 10 chambers. The bottom panel shows a chamberin which no HyHel5 cells are loaded. The locations of the loaded HyHel5cells were recorded, once loaded into the device. Referring to FIG. 74B,the 4B2 cells were loaded into the chambers at an average ratio of thetwo cell lines loaded into the device at an average of 25 cells perchamber, corresponding to 250 times the concentration of HyHel5 cells.

The device chambers were then washed and beads functionalized withrabbit polyclonal anti-mouse IgG antibodies (readout particles) wereloaded into the chambers. The chambers were then isolated and incubated,resulting in the capture of the antibodies generated by the cells ineach chamber on the beads in that chamber. Referring to FIG. 74C,fluorescently labeled HEL (10 nM) was included in the medium during theincubation step and fluorescent images were taken to identify thechambers having beads with bound HyHel5 and to monitor the accumulationof HyHel5 antibodies on the beads. As can be seen in FIG. 74C,fluorescence was only detected in the top panel, indicating that onlychambers having HyHel5 cells generate strong fluorescent signal on beadswhen incubated with the fluorescently labeled HEL antigen. Referring toFIG. 74D, the device was then incubated with fluorescently labeledpolyclonal anti-IgG antibodies to identify chambers having beads withbound IgG, either secreted from HyHel5 cells or 4B2 cells. All chambersgenerated fluorescent signal when exposed to fluorescently labeledanti-IgG antibody, indicating that antibodies from both cell lines werecaptured in all chambers. Following incubation with fluorescentlylabeled HEL, the chambers were flushed with medium in the absence oflabeled HEL antigen, and images were taken to monitor the dissociationkinetics of the HyHel5-HEL binding in the chambers, as shown in FIG.74E. FIG. 74F shows the fluorescence over time of 600 chamberscontaining a mix of HyHEL5 and 4B2 hybridoma cells as described above,incubated in the presence of Protein A beads and 10 nM lysozyme.Chambers containing HyHEL5 cells were easily distinguishable by theirfluorescence above the baseline, demonstrating the ability of thissystem to detect rare cells secreting an antigen-specific mAb.

In a different example, a population of HyHEL5 hybridoma cells secretingantibodies against hen-egg lysozyme (HEL) was loaded in the microfluidicdevice at limiting dilution. The device was imaged to determine thepresence of HyHEL5 cells in each of the chambers. DMS-1 hybridoma cellssecreting antibodies that do not bind HEL were subsequently loaded at anaverage concentration of approximately 5 cells per chamber in the samearray. Cells were incubated in the presence of readout antibody-capturebeads (Protein A beads coated with rabbit anti-mouse antibody) and thesecretion of lysozyme-specific antibodies was measured by washing with a1 nM solution of lysozyme labeled with Alexa-488. Bright field imagesobtained after loading HyHeL5 cells (top), bright field images takenafter incubation with DMS-1 cells and beads (middle), and correspondingfluorescent images (bottom) are shown for three representative chamberscontaining DMS-1 cells only (FIG. 75A) or a mixture consisting of asingle HyHEL5 cell and multiple DMS-1 cells (FIG. 75B). The distributionof fluorescence intensity is shown for chambers containing DMS-1 cellsonly (FIG. 75C) or both HyHEL5 cells and DMS-1 cells (FIG. 75D). Cellssecreting antibodies against hen-egg lysozyme in the HyHEL5 populationwas detected even in the presence of multiple DMS-1 cells secretingnon-antigen-specific antibodies.

Example 7—Selection of mAbs from Single Cells Using Bead- and Cell-BasedBinding Assays

Single hybridoma effector cells (4B2) were loaded into individualchambers of a microfluidic device and binding of secreted antibodies(IgG against anti-human CD45) was measured using both a bead-based andcell-based readout binding assays. Readout cells in this experiment wereK562 cells endogenously expressing the human CD45 membrane receptor andstained with carboxyfluorescein succinimidyl ester (CFSE) prior tofixation so as to distinguish them from the effector cells. Singleeffector cells were provided to individual chambers of the device andincubated with a plurality of readout K562 cells overnight. Next, theeffector cells were incubated with readout Protein A beads for anadditional 2 hours and then stained with a detection antibody(fluorescently labeled anti-mouse IgG), which binds the secreted IgGanti-human CD45 antibody. Fluorescence of individual chambers was thenmeasured. A schematic of the experiment is provided in FIG. 76A

FIG. 76B is a graph of mean fluorescence intensity of readout cells andreadout beads measured by automated image analysis for empty chambersand chambers containing a single hybridoma effector cell. FIG. 76C (leftto right) shows a single chamber with a single hybridoma cell (leftpanel). The hybridoma cell in this example divided overnight. Theremaining panels show fluorescent and merged images of anti-CD45antibody staining in the same chamber following an overnight incubationwith target fixed K562 cells and a 2-hour incubation period with proteinA beads (labeled with appropriate polyclonal anti-mouse IgG antibodies).A person skilled in the art will understand that this assay can bemodified to be performed with live readout cells and/or differentspecies of readout cells.

Example 8—Cell-Based Immunization and Cell Binding Assays

Mice were immunized with fixed cells from a human ovarian cancer cellline (TOV21G) (FIG. 77A). Antibody-secreting effector cells were sortedusing FACS and were then injected in the microfluidic device andincubated with readout cells (fixed and live TOV21G cells) stained withCFSE. Antibody binding was visualized using a secondary labeledantibody. FIG. 77B shows the results of this experiment. From left toright: plasma and readout cells (live and fixed) after loading on chip.Readout cells were stained with CFSE for identification. Antibodybinding on the cell surface of live and fixed cells was visualized witha secondary labeled antibody (anti-mouse IgG). Far right shows anegative chamber (no effector cells) with very low signal on the readoutcells.

Example 9—Maintenance of Cellular Viability and Secretion

Conditions were optimized for maintaining cellular viability andsecretion of ASC effector cells for 5-8 days. FIG. 78 shows cellsurvival and antibody-secretion by ELISPOT from mouse ASCs grown for 8days (FIG. 78A), and human ASCs (FIG. 78B) grown for 5 days in completeRPMI with IL-6 10 ng/mL.

For immunization procedures, mice were subcutaneously injected with henegg-white lysozyme and Alhydrogel adjuvant (Accurate Chemical &Scientific Corporation) 3 times at 2 week intervals. Humans wereimmunized with influenza vaccine 1 week prior to blood collection.Immunization procedures were performed in accordance with approvedprotocols by the University of British Columbia. Mouse spleen cells wereisolated and stained with PE anti-mouse CD138 (BD Pharmingen) and sortedby FACS for CD138+ cell population (see Example 10 for a furtherdescription). Human peripheral blood mononuclear cells (PBMCs) wereisolated and stained with markers as described in Wrammert et al.(2008), Nature Letters 453, pp. 667-671, the disclosure of which isincorporated by reference in its entirety for all purposes.

Enzyme-Linked-Immunospot (ELISPOT) assay was performed in polyvinylidenefluoride (PVDF) membrane-lined 96-well microplates (Millipore). The PVDFmembrane was pre-wet with 70% ethanol and washed with PBS. Plates werecoated overnight with goat anti-mouse IgG (H+L) (Jackson Immunoresearch)or rabbit anti-human IgG (H+L) (Jackson Immunoresearch antibody(1:1000/100 pt/well) and washed 3-4 times with PBS. Cells were thenadded and incubated for 20 h at 37° C. Cells were removed by washing 3-4times with PBS containing 0.1% Tween (PBS-Tween), and plates wereincubated with alkaline phosphatase-goat anti-mouse IgG (H+L) (JacksonImmunoresearch) (1:1000/100 uL/well). After washing 3-4 times withPBS-Tween, plates were incubated with BCIP/NBT chromogenic agent(Sigma-Aldrich B6404-100ML). Spots were counted with the aid of anupright microscope and CCD camera.

Example 10—Methods for Enriching Plasma Cells

Antibody-secreting cells were enriched in species with known markers,specifically CD138 plasma cell marker in mice (FIG. 79). First, micewere immunized intraperitoneally with 5×10⁶ ovarian carcinoma cells(TOV-21G) 3 times at one week intervals, in accordance with approvedanimal care protocols. Mouse spleen cells were stained with PEanti-mouse CD138 (BD Pharmingen) and sorted by FACS for CD138+, andCD138− as a negative control (FIG. 79A). ELISPOT was performed asdescribed in Example 9 to detect antibody-secretion from the sorted andunsorted cell populations (FIG. 79B). A 20-fold increase in ASCs fromthe CD138+ population compared to unsorted spleen cells was observed(FIG. 79C). Although not performed here, enrichment can also be doneusing commercially available magnetic-based enrichment kits (e.g.,StemCell Technologies, Miltenyi) alone or in combination with FACSsorting.

Antibody-secreting cells were also enriched from species lackingestablished markers for plasma cells by using the ER-Tracker™ (LifeTechnologies) in rabbits (FIG. 80). Rabbits were immunized withinfluenza vaccine intradermally 4 times at 1 week intervals, followed byboosts 1 week prior to blood collection, in accordance with approvedanimal care protocols. Rabbit PBMCs were stained with ER-Tracker (LifeTechnologies) and mouse anti-rabbit IgG (Jackson Immunoresearch), andsorted by FACS for ER^(high)IgG^(low) population (FIG. 80A). ELISPOT wasperformed as described in Example 9 to detect antibody-secretion fromthe ER^(high)IgG^(low) population and unsorted PBMCs control (FIG. 80B).A 6-fold increase in antibody secretion from the ER^(high)IgG^(low)population compared to unsorted PBMCs was observed (FIG. 80C).

Example 11—Enrichment Using Influenza Human Systems

Peripheral blood mononuclear cells (PBMCs) were isolated from the bloodof a human patient 7 days following immunization with the influenzatrivalent vaccine. Cells were enriched based on CD138 expression using acommercially available bead separation kit (Stem Cell Technologies) andloaded in a microfluidic device at an average density of 11 cells perchamber (i.e., heterogeneous cell populations comprised on average 11cells) for a total of approximately 44,000 cells. Cells were incubatedwith Protein A beads for 2 hours, and with fluorescently labeled H1N1and H3N2 in different colors. A total of 171 H1N1-positive chambers and199 H3N2-positive chambers (e.g., FIG. 81A) representingantigen-specific frequencies of 0.39% and 0.45%, respectively. Thecontents of 24 of these chambers were recovered and cultured overnightin a multiwell plate (FIG. 81B) and then introduced in a secondmicrofluidic device the next day. After enrichment, the frequencies ofH1N1-positive H3N2-positive chambers (FIG. 81C) were 7% and 6%,respectively, meaning that a 13- to 18-fold enrichment was obtained(FIG. 81D).

Example 12—Bead Aggregation as an Indicator of Antibody Secretion

Chambers of a microfluidic device were loaded with a mixture of cellssecreting human antibodies and non-antibody secreting cells. Cells wereincubated for 2 hours in the presence of protein-A beads and thenstained using secondary labelled antibodies. The beads in chambers thatcontained antibody-secreting cells formed aggregates (FIG. 82) whilebeads remained dispersed in the absence of secreted antibodies (FIG.83).

Example 13—Affinity Measurements for HEL-Specific Hybridomas

A population of hybridoma effector cells (HyHEL5) producing an antibodywith high affinity for hen-egg lysozyme (HEL) was first introduced in amicrofluidic device at limiting dilution. A picture set was taken toidentify chambers containing HyHEL 5 cells, and then a second populationof hybridoma cells (D1.3) secreting an antibody with low affinity forHEL was loaded in the same device. A second picture set was taken andchambers containing either one HyHEL5 cell or one D1.3 cell wereidentified. Protein A beads coated with a rabbit anti-mouse antibodywere introduced in the chambers and incubated with the cells for 2hours. At the end of the incubation period, labeled antigen wasintroduced in the device at increasing concentrations between 100 fM and10 nM in a step-wise fashion. FIG. 84A shows a diagram of thisexperiment, and micrographs of the chambers with differentconcentrations are shown in FIG. 84B. The bead fluorescence intensitywas measured at every step, normalized to the background for everychamber. Chambers containing HyHEL5 were distinguished from thosecontaining D1.3 cells based on the affinity measurements (FIG. 84C).Example curves from representative chambers containing single cellsHyHEL5 and D1.3 and the images from these same chambers are shown inFIG. 84D and FIG. 84B, respectively. Affinities for HyHel5 and D1.3antibodies are 30 μM and 1.5 nM, respectively (Singhal et al. (2010),Anal Chem 82, pp. 8671-8679, in corporate by reference herein for allpurposes).

Example 14—Identification of Antigen-Specific Cells with or withoutChamber Isolation

Human plasma cells were enriched from PBMCs obtained from the blood ofhuman patients one week after immunization with the trivalent influenzavaccine. Cells were introduced in two different microfluidic devices andincubated in the presence of Protein A beads (readout beads) for 2hours. In one device (FIG. 85), individual chambers comprisingheterogeneous cell populations and readout beads were isolated from oneanother with a valve during the incubation. In the second device (FIG.86), the valve was left open. Labeled antigens (H1N1 and H3N2) wereintroduced in the device at the end of the incubation period for 15minutes and then the chambers were washed with media. FIG. 85 and FIG.86 show a plurality of chambers from a section of each array, withH3N2-positive hits identified by white arrow. In the absence of mainvalve isolation (FIG. 86), the background in surrounding chambers washigher than with the main valve closed (FIG. 85). Howeverantibody-carryover did not prevent clear distinction between negativeand positive chambers. The direction of the flow channels is indicatedby black arrows.

Example 15—Selection of Novel Mouse Antibody Secreting Cells Based onAffinity Measurements

Plasma cells were isolated from the bone marrow of a mouse immunizedwith hen-egg lysozyme. Approximately 23,800 cells were distributedacross 3 sub-arrays containing 6,144 chambers in a microfluidic device(average density: ˜4 cells/chamber). Cells were incubated with Protein Abeads coated with a rabbit anti-mouse capture antibody for 2 hours,washed with 10 nM of labeled hen-egg lysozyme and imaged. 117antigen-specific chambers were identified, the contents of which wererecovered with a microcapillary and reinjected at limiting dilution in afourth sub-array containing 2,048 chambers. The reinjected cells wereincubated with Protein A beads coated with a rabbit anti-mouse captureantibody for 2 hours, and then exposed to increasing concentrations oflabeled antigen. Images were taken at each step and the beadfluorescence intensity was measured. FIG. 87 shows an example of bindingcurves from 2 single cells (labeled Mm20 and Mm25) secreting antibodieswith different affinities. These cells were recovered as follows.

117 chambers containing a total of 882 cells were identified ascontaining at least one antibody-secreting cell. The contents of all 117chambers were recovered with a microcapillary, pooled and immediatelyre-injected into the remaining 2,048 empty chambers of the originaldevice using the recovery robot shown in FIG. 58. FIG. 98 shows apicture of the microcapillary in proximity of the injection portimmediately before reinjecting the cells. The recovery process took atotal of 1 hour and 35 minutes, or 49 seconds per chamber. Afterre-injection, a total of 682 cells were present in the analysischambers, which represented 77% of the initial population recovered (882cells).

The re-injected cells were immediately assayed again for the presence ofone or more antibody secreting cells. 38 chambers were identified ascontaining at least one antibody-secreting cell (38/117=32%). Of these,38 chambers, 19 contained single cells (16% of 117). The contents of all38 chambers, along with 10 control chambers (5 no-cell and 5non-secreting controls) were recovered for RT-PCR. The recovery processtook a total of 2 hours and 15 minutes, or 170 seconds per chamber. In43 of 48 samples (90%), all cells in the chambers were visually verifiedto have been recovered by the capillary. In 5 of 48 samples, at leastone cell was seen to stick to the chamber bottom and was not recovered.

The following sequences were retrieved.

Mm20 (IGHV1-9*01) IGHD2-4*01, IGHD2-4*01,IGHD2-9*02 IGHJ2*01 heavy chain nucleic acid sequence (SEQ ID NO: 1): ATGGAATGGACCTGGGTCTTTCTCTTCCTCCTGTCAGTAACTGCAGGTGTCCACTCCCAGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCAACTGGCTACACATTCAGAAACTACTGGATAGAGTGGATAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGAAAGTGGTAGTATTAATTACAATGAGAAATTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACTTGCAACTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTATTGTTTTTATGATAATTACGTTTTTGACTACTGGGGCCAaggcacCACTctcACMm20 light chain amino acid sequence (SEQ ID NO: 2): M E W T W V F L F L L S V T A G V H S Q V Q L Q QS G P E L M K P G A S V K I S C K A T G Y T F R NY W I E W I K Q R P G H G L E W I G E I L P E S GS I N Y N E K F K G K A T F T A D T S S N T A Y LQ L R S L T S E D S A V Y Y C F Y D N Y V F D Y W G Q G T T LMm20 (IGKV4-59*01) IGKJ5*01 light chain nucleicacid sequence (SEQ ID NO: 3): ATGGATTCTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCGGTCATACTATCCAGTGGACAAATTGTTCTCATCCAGTCTCCAACAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAACTCAAGTTTCAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATTAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAGCAGTGGAGTAGAAACCCCACGTTCGGTGCtggacCaAGCtGa Mm20 light chain amino acid sequence(SEQ ID NO: 4):  M D S Q V Q I F S F L L I S A S V I L S S G Q I VL I Q S P T I M S A S P G E K V T M T C S A N S SF S Y M H W Y Q Q K S G T S P K R W I Y D T S K LA S G V P A R F S G S G S G T S Y S L T I S S M EA E D A A T Y Y C Q Q W S R N P T F G A G T K L >Mm25 (IGHV2-9-1*01) IGHD2-3*01 IGHJ3*01heavy chain nucleic acid sequence (SEQ ID NO: 5): ATGCAAGCAGTGGTATCAACGCAGAGTACGGGGAAGGAGTCAGGACCTGGCTTGGTGGCGCCCTCACAGAGCATGTCCATCATGTGCACTGTCTCTGGGTTTTCATTAAGCAACTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATTTGGGCTGGTGGAAACACAAATTATAATTCGGCTCTCATGTCCAGACTGAGCATCAGCAAAGACAAGTCCAAGAGTCAAGTTTTCTTAAAAATGAACCGTCTGGAAACTGATGACACAGCCATGTACTATCTGTGCCAGTGTAGGATGGTTACCCCTTGCTTACTGGGCCAAGGMm25 heavy chain amino acid sequence (SEQ ID NO: 6): M Q A V V S T Q S T G K E S G P G L V A P S Q S MS I M C T V S G F S L S N Y G V H W V R Q P P G KG L E W L G V I W A G G N T N Y N S A L M S R L SI S K D K S K S Q V F L K M N R L E T D D T A M YY L C Q C R M V T P C L L G Q >Mm25 (IGKV6-17*01) IGKJ4*01 light chainnucleic acid sequence (SEQ ID NO: 7): ATGGAGTCACAGATTCAGGTCTTTGTATTCGTGTTTCTCTGGTTGTCTGGTGTTGACGGAGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTATTTCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCCGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCIITCAC GTCGGCTcggGaCAAagTGMm25 light chain amino acid sequence (SEQ ID NO: 8): M E S Q I Q V F V F V F L W L S G V D G D I V M TQ S H K F M S T S V G D R V S I T C K A S Q D V SI S V A W Y Q Q K P G Q S P K L L I Y S A S Y R YT G V P D R F T G S G S G T D F T F T I R S V Q A E D L A V Y Y C Q Q H Y S T P F T S A R D K V

Example 16—K_(off) Values for HyHEL5 Hybridoma Cells

A mixture of HyHEL5 hybridoma (low K_(off)) and a background of D1.3hybridoma (high K_(off)) cells were screened in a microfluidic deviceaccording to an embodiment of the invention. Chambers in which HyHEL5hybridoma were present were detected by fluorescence intensityassociated with Ab secretion (i.e., fluorescence accumulation on beads)before and antigen release (for K_(off) measurements) following a washstep for each chamber. FIG. 88 shows a bar graph of the remainingfluorescence level of beads in HyHEL5-positive wells is higher than inthe rest of the wells at the end of the wash.

Example 17—Detection of Rare Circulating Antibody-Secreting Cells inHumans Against Specific Antigens

In one embodiment, the invention is used to screen for extremely rarecells, for instance antigen-specific plasma B cells that occur at basallevel, i.e., without recent immunization or infection with the antigen.FIG. 89A shows a population of heterogeneous cells containing enriched Bcells and erythrocytes from a healthy patient. The population containedat least one antibody secreting cell as measured by whole IgG stainingon readout bead particles (FIGS. 89B and 89C). In addition, thispopulation contained at least one effector cell secreting antibodiesspecific to H1N1 as measured by the binding of a labeled H1N1 antigen tothe readout beads that had captured the antibody (FIGS. 89D and 89E).The entire screen was performed using ˜30 cells per chamber in an arrayof 1,600 chambers for a total population of 48,000 cells. This allowedthe detection of one effector cell present at a frequency of 0.000625%.The cells of the chamber were recovered (FIG. 89F) and the heavy andlight chains from that effector cell were amplified.

This assay can also be performed to find more abundant cells, forinstance after a natural infection or about one week post-immunizationwith an antigen.

Example 18—Simultaneous Analysis of Extracellular and IntracellularBiomolecules

The analysis of extracellular and intracellular components was assessedin the context of a cell signaling readout assay. CHO cells engineeredto overexpress the GPCR CXCR4 with beta-galactosidase fragmentcomplementation (DiscoverX) were used to quantify the binding of theligand CCL22 to the receptor. Activation of the receptor by the ligandcaused intracellular beta-galactosidate complementation, which was thenmeasured by lysing the cells in the presence of a beta-galactosidatesubstrate that yields a chemiluminescent signal. An example of thisassay performed in multiwell plate is shown in FIG. 90. The assay wasperformed following the protocol provided by the manufacturer(DiscoverX). Three different concentrations for the agonist/ligand CCL22(100 nM, 1 nM, 0.01 nM) were incubated with the cells before adding thelysis/chemilumiscent reagents. Light intensity chemiluminescent signalwas acquired using a Tecan M200 plate reader from each wellcorresponding to a particular condition.

This assay can be used in conjunction with a cell binding assay (e.g.,the experiment shown at FIG. 77) to find antagonist antibodies specificfor a cell surface receptor. Implementation of this lysis assay in themicrofluidic chamber requires segregation of the effector cell from thereadout cell(s) if it is desired to recover a live, viable effectorcell. Such an assay could be implemented in a device such as the onepresented in Examples 1 and 2.

Example 19—Antibody Screening and Sequence Recovery from Human Cells

Blood was collected 7 days after immunization from patients that hadreceived the seasonal flu vaccine (FIG. 91). PBMCs were isolated using acommercially available kit (SepMate, StemCell Technologies) and plasmacells were enriched using FACS based on the markers described inWrammert et al. (2008), Nature Letters, 453:667-671, incorporated byreference herein in its entirety. Cells were then loaded in microfluidicdevices at limiting dilution and incubated for 2 hours with Protein Amicrospheres (4.9 μm in diameter, Bang Laboratories). H1N1 and H3N2labeled antigens (two different colors: 488 nm and 594 nm emission,respectively) were introduced in the array, incubated for 15 minuteswith the plasma cells, and the contents of microfluidic chambers werewashed with media before imaging. After detection of chambers containingantigen-specific antibodies, labeled anti-human antibody (Dylight 594)was introduced in the device, incubated for 15 minutes, and then washedto determine the overall frequency of IgG secreting cells. An example ofa single plasma cell cross-reactive for both H1N1 and H3N2 is shown inFIG. 92. Antigen-specific cells were either recovered immediately orcultured overnight in the microfluidic devices for next-day recovery.Cells remained viable and in some cases underwent division (e.g., FIG.93).

Referring to FIG. 94, cells from eight chambers positive for H1N1 and/orH3N2 (flu specific), and IgG (i.e., not antigen-specific) were recoveredand lysed. The RNA from the lysed cells was subjected to reversetranscription followed by antibody heavy and light chain (Kappa/lambda)specific PCR. Four samples out of eight provided a positive PCRamplification for both heavy and light chains (50% efficiency with 100%pairing), with an expected product size of ˜400 bp, namely samples 3(H1N1 positive), 4 (H1N1/H3N2 positive), 5 (H1N1/H3N2 positive), and 6(IgG positive, but not antigen specific). The nucleic acid ladder shows100 base pair increments. Sanger sequencing from these purified PCRproducts confirmed that the sequences were specific for heavy, kappa andlambda human immunoglobulin.

Referring to FIG. 95, sequences of two human mAbs (Hs-7 and Hs-15) thatwere amplified from single cells obtained from patients immunized withseasonal flu vaccine. Cells were selected in a microfluidic assay basedon their ability to secrete mAbs having a cross-reactivity tohemagglutinin subtypes H1N1 and H3N2. Antibodies sequences wereretrieved by RT-PCR and sequencing as described herein (FIG. 95A). BothmAbs belong to the same clonotype as apparent by shared gene usage, CDRlength and junctional sequences. Mutations are shown in lighter grey(FIG. 95B). Antibody sequences were cloned and expressed in HEK293 cellsto validate their binding properties. Protein A beads were incubatedwith the cell supernatant for 3 hours, washed, incubated with eitherlabelled H1N1 or H3N2 at different concentrations and then imaged tomeasure the bead fluorescent intensity. Both mAbs cross-reacted asexpected but, consistent with single cell measurements, had differentaffinities for H1N1 and H3N2 (FIG. 95C).

Hs7 (IGHV4-39*01) IGHD3-3*02 IGHJ3*02 Heavychain nucleic acid sequence (SEQ ID NO: 9): ATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCTCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGTCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGACTCCATCACCAGCAGTACTTACGACTGGGGCTGGATCCGTCAGCCCCCCGGGAAGGGCCTGGAGTGGATTGGCAATGTCTATTATAGAGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACAGGTCCAGGACCCAGATCTCCCTGAGGCTGAGCTCTGTGACCGCCGCTGACACGGCTCTGTATTTCTGTGCGAGACACCCGAAACGTCTAACGGTTTTTGAAGTGGTCAACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTTTTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCTGCTGACGAGGCACTGAGGACTGHs7 Heavy chain amino acid sequence (SEQ ID NO: 10): M K H L W F F L L L V A A P R W V L S Q V Q L Q ES G P G L V K S S E T L S L T C T V S G D S I T SS T Y D W G W I R Q P P G K G L E W I G N V Y Y RG S T Y Y N P S L K S R V T I S V D R S R T Q I SL R L S S V T A A D T A L Y F C A R H P K R L T VF E V V N A F D I W G Q G Q T M V T V F S A S T KG P S V F P L A P S S K S T S G G T AHs7 (IGLV2-14*01) IGLJ3*02 Light chainnucleic acid sequence (SEQ ID NO: 11): ATGGCCTGGGCTCTGCTACTCCTCACCCTCCTCACTCAGGGCACAGGGTCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGCATCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAATCAGCAGTGACATTGGTGGTTATAGCTCTGTCTCCTGGTACCAAGCGCACCCAGGCAAAGCCCCCAAACTCATGATCTATGATGTCAATAATCGGCCCTCAGGCATTTCTAATCGCTTCTCTGGTTCCAAGTCTGGCAACACGGCCTCCCTGGCCATCTCTGGGCTCCAGgctgaGGACGAGGCAGATTATTACTGCAGCTTATATACAAGTATCAACGCTTCCATAGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAA GCCAACAAGGCCACAHs7 Light chain amino acid sequence (SEQ ID NO: 12): M A W A L L L L T L L T Q G T G S W A Q S A L T QP A S V S A S P G Q S I T I S C T G I S S D I G GY S S V S W Y Q A H P G K A P K L M I Y D V N N RP S G I S N R F S G S K S G N T A S L A I S G L QA E D E A D Y Y C S L Y T S I N A S I V F G G G TK L T V L G Q P K A A P S V T L F P P S SHs15 (IGHV4-39*01) IGHD3-3*01 IGHJ3*02 Heavychain nucleic acid sequence (SEQ ID NO: 13): ATGAAGCACCTGTGGTTCTTCCTTCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCCCAGCTGCAACTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGACTCCATCAGCAGTAGTACTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGAAAGGGGCTGGAGTGGATTGCCTTTATCTTTTATAGCGGGAGCACCTTCTACAACCCGTCCCTCAAGAGTCGAGTCACCGTCTCCGTAGACAGGTCCACGAACCAGTTCTCCCTGAGGCTGAAGTCTGTGACCGCCGCAGACACGTCCAGATATTACTGTGCGAGACACCCAAAACGTATCTCGATTTTTGAAGTGGTCAACGCTTTTGATATCtGGGGCCAGGGGACAATGGTCACCGTCTCTTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCTGCTGACGAGGCACTGAGGACTGHs15 Heavy chain amino acid sequence (SEQ ID NO: 14): M K H L W F F L L L V A A P R W V L S Q L Q L Q ES G P G L V K P S E T L S L T C T V S G D S I S SS T Y Y W G W I R Q P P G K G L E W I A F I F Y SG S T F Y N P S L K S R V T V S V D R S T N Q F SL R L K S V T A A D T S R Y Y C A R H P K R I S IF E V V N A F D I W G Q G T M V T V S S A S T K GP S V F P L A P S S K S T S G G T A >Hs15 (IGLV2-14*01) IGLJ2*01 IGLJ3*01 Lightchain nucleic acid sequence (SEQ ID NO: 15): ATGGCCTGGACTCTGCTATTCCTCACCCTCCTCACTCAGGGCACAGGGTCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCACCTGCACTGGAATCAGCAGTGACGTTGGTGCTTATAATTCTGTCTCCTGGTACCAGCAGTACCCAGGCAAATCCCCCAAGCTCATGATTTATGATGTCAGTAATCGGTCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGACAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTTCTTATTTCTGCAGCTTATATAGAAGCAGCACCACTTCCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTACGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAA GCCAACAAGGCCACAHs15 Light chain nucleic acid sequence (SEQ ID NO: 16): M A W T L L F L T L L T Q G T G S W A Q S A L T QP A S V S G S P G Q S I T I T C T G I S S D V G AY N S V S W Y Q Q Y P G K S P K L M I Y D V S N RS S G V S N R F S G S K S D N T A S L T I S G L QA E D E A S Y F C S L Y R S S T T S V V F G G G TK L T V L R Q P K A A P S V T L F P P S S

Example 20—Antibody Screening and Sequence Recovery from a HeterogeneousPopulation of Rabbit Cells

Rabbits were immunized with the seasonal flu vaccine. Peripheral bloodmononuclear cells (PBMCs) were recovered and either screened directly orsorted with flow cytometry to enrich for plasma cells. The cells wereinjected in the microfluidic device and assayed for H1N1 and H3N2specificity. Protein A-coated beads used for fluorescence-baseddetection were also visible. Cells were incubated with beads for 2hours, then fluorescently labeled antigen was introduced for 15 minutesand unbound antigen was washed away with media. FIGS. 96A and 96B showexamples of H1N1- and H3N2-positive chambers, respectively.

FIG. 97A shows bright field images of several rabbit cells in fourmicrofluidic chambers that have been screened for antigen-specificsignal. FIG. 97B shows H1N1 antigen specific antibody screening fromrabbit antibody producing cells. The signal was very low and thereforeno cell seemed to express H1N1 specific antibodies. FIG. 97C shows H3N2antigen specific antibody binding from rabbit antibody producing cells.Signal was variable from one chamber to another, however all chambersare positive for H3N2 binding. After cell recovery from the microfluidicchambers using an automated robot and microcapillary, heavy and lightchain antibody specific polymerase chain reaction (PCR) products wereran on a 2% Egel (Invitrogen). Bands (˜400/500 bp) were visible for 3out of 4 heavy chains and 4 out of 4 light chains for the 4 samplestested (FIG. 97D). Sanger sequencing revealed that two heavy chainsequences were rabbit heavy chains (world-wide-website: imgt.org)whereas one contains multiple peaks suggesting that more than one cellin this chamber might have been an antibody-secreting cell. Two lightchains out of four were determined to be rabbit light chains while thetwo others contained multiple peaks. Paired heavy and light chains fromchambers 1 and 3 showed that it is possible to determine the sequence ofa single effector cell from a heterogeneous cell population. Thesequences recovered for the variable regions having single peaks were asfollows:

Heavy 1 (SEQ ID NO: 17): GCTAGCCACCATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCATAGTCTCTGGAATCGACCTCAGTAGCTATGCAATGGGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCTGGAATACATCGGAATCATTAGTAGCAGTGGTATCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGACAATCACCGATCTGCAACCTTCAGACACGGGCACCTATTTCTGTGCCAGAGGGTCTCGTTATAGTGCTTTTGGTGCTTTTGATACCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGCAAGCTTNAN Heavy 3 (SEQ ID NO: 18): TTTGGCTAGCCACCATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTATGCAATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTAATAATAATGGTGACACATACTACGCGAGCTGGCCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGATCGTGGTAATAGTTATTACTTTGGATTGGACTACTTTAACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGCAAGCTTTATAN Light 1 (SEQ ID NO: 19): CTCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCCTTCGAATTGACCCAGACTCCATCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGTATTAATAGTTGGTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCATCCAATCTGGCATCTGGGGTCCCATCGCGGTTCAGAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGNCGATGCTGCCACTTACTACTGTCAAAGCNATTATGCTACTAGTAGTGTTGATTATNATGCTTTCGGCGGAAGGACCGAGGTGGTGGTCAANACTGCGGCNGTANTANTNNN Light 3 (SEQ ID NO: 20): TGCAGCTAGCCACCATGGACACGAGGGCCCCCATCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCAGGTGTGCCCTTGTGATGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTGATAGTTGGTTATCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAGGCTCCTGATCTATTATGCATCCAATCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAATACACTCTCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAAGAGGGTTATAGTAGTGGTAATGTTGATAATGTTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAACTGCGNCCGCTATAN

Example 21—Expression and Validation of Antibodies from Human Sequences

The expression of a human anti-MCP1 antibody was carried out in HEK293cells using liposome-based transfection. Expressed antibody wasprecipitated from growth media on Protein A-coated beads and tested withfluorescently labeled MCP-1 antigen. The affinity of recombinantantibody was compared to the antibody of the same amino-acid sequenceproduced by the commercially available stable CHO cell line (ATCC®PTA-5308′^(M)) (FIG. 99).

Clone Hu-11K2-3f2-H2. Heavy and light chain variable sequences weresynthesized and cloned into pFUSEss-CHIg-hG1 and pFUSE2-CLIg-hkexpression vectors respectively.

aMCP1-Heavy (SEQ ID NO: 21):GAATTCCATGCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCCGGCAGCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCCTGACCATCAGCGACACCTACATGCACTGGGTGCGCCAGGCTCCAGGCCAGGGACTGGAATGGATGGGCAGAATCGACCCCGCCAACGGCAACACCAAGTTCGACCCCAAGTTCCAGGGCAGAGTGACCATCACCGCCGACACCAGCACCTCCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCGTGTTCGGCTTCTTCGACTACTGGGGCCAGGGCACCACCGTGA CCGTGTCATCTGCTAGCaMCP1-Light (SEQ ID NO: 22):ACCGGTGCCACCATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGTCTCTGGCCCTCGTGACGAATTCAGCCATGGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACATGCAAGGCCACCGAGGACATCTACAACCGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATTAGCGGAGCCACCAGCCTGGAAACCGGCGTGCCAAGCAGATTTTCCGGCAGCGGCTCCGGCAAGGACTACACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTTTTGGAGCGCCCCCTACACCTTTGGCGGAGGCACCAAGGTGGAAATCAAGC GTACG

Example 22—Mouse Sequence Recovery from Single Cells Followed by Cloningand Expression of Antibodies for Validation

D1.3 and HyHEL5 were loaded sequentially at limiting dilution in amicrofluidic device. A picture set was taken after introducing each celltype in the device to record the position of D1.3 and HyHEL5 cells.Cells were incubated with Protein-A beads coated with rabbit anti-mousecapture antibodies and 10 nM of labeled hen-egg lysozyme for 2 hours.Single antibody-secreting cells were recovered with a microcapillarycontrolled by a robot, transferred into a tube for RT-PCR, and then theantibody sequences were recovered. Control chambers were recoveredbetween each single cell as negative control. FIG. 100 shows a gel heavyand light chains from 2 single cells, with no signal in the controls.Sequences of variable regions of heavy and light chains of HyHEL5 (highaffinity) and D1.3 (low affinity) antibodies were synthesized and clonedinto pFUSEss-CHIg-mG1 and pFUSE2ss-CLIg-mk expression vectors.Antibodies were produced in HEK293 cell line, captured on ProteinA-coated beads covered with Rabbit anti-mouse antibody. The affinity wastested using increasing concentrations of fluorescently labeled lysozyme(FIG. 101).

D1.3-Heavy (SEQ ID NO: 23): ATGCAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTCTCATTAACCGGCTATGGTGTAAACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAATGATTTGGGGTGATGGAAACACAGACTATAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCACACTGATGACACAGCCAGGTACTACTGTGCCAGAGAGAGAGATTATAGGCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTC AGCTAGCD1.3-Light (SEQ ID NO: 24): ATGGACATCCAGATGACTCAGTCTCCAGCCTCCCTTTCTGCGTCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGGGAATATTCACAATTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATTATACAACAACCTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGAGTACTCCTCGGACGTTCGGT GGAGGCACCAAGCTCGAGHyHEL5-Heavy (SEQ ID NO: 25): ATGGAGGTCCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCAGGGGCCTCAGTGAAGATATCCTGCAAAGCTTCTGGCTACACATTCAGTGACTACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGTAGCACTAATTACCATGAGAGATTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAGCACAGCCTACATGCAACTCAACAGCCTGACATCTGAAGACTCTGGCGTCTATTACTGCCTCCATGGTAACTACGACTTTGACGGCTGGGGCCAAGGCACCACTCTCACAGTCTCCTC AGCTAGCHyHEL5-Light (SEQ ID NO: 26): ATGGATATCGTTCTCACACAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGTTCAAGTGTAAATTACATGTACTGGTACCAGCAGAAGTCAGGCACTTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGACTGAAGATGCTGCCACTTATTACTGCCAACAGTGGGGTCGTAACCCCACGTTCGGAGGGGGG ACCAAGCTCGAG

Example 23—Validation of Novel Mouse Antibody Sequences Obtained fromMicrofluidic Screening

Splenocytes were isolated from a mouse immunized with hen-egg lysozymeand screened for their antibody secretion in a microfluidic device. Thesequence from the antigen-specific cell R05C14 was recovered, cloned andexpressed in HEK293 cells. Antigen binding was confirmed by capturingthe antibody on Protein A beads coated with rabbit anti-mouse antibody,incubating with 10 nM labeled hen-egg lysozyme and measuring thefluorescence intensity (FIG. 102).

R05C14-Heavy variable (SEQ ID NO: 27): TGGAAGGTGGTGCACACTGCTGGACAGGGATCCAGAGTTCCAGGTCACTGTCACTGGCTCAGGGAAATAGCCCTTGACCAGGCATCCCAGGGTCACCATGGAGTTAGTTTGGGCAGCAGATCCAGGGGCCAGTGGATAGACAGATGGGGGTGTCGTTTTGGCTGAGGAGACTGTGAGAGTGGTGCCTTGGCCCCAGCAGTCCCCGTCCCAGTTTGCACAGTAATATGTGGCTGTGTCCTCAGGAGTCACAGAAATCAACTGCAGGTAGCACTGGTTCTTGGATGTGTCTCGAGTGATAGAGATTCGACCTTTGAGAGATGGATTGTAGTAAGTGCTACCACTGTAGCTTATGTACCCCATATACTCAAGTTTGTTCCCTGGGAATTTCCGGATCCAGCTCCAGTAATCATTGGTGATGGAGTCGCCAGTGACAGAACAGGTGAGGGACAGAGTCTGAGAAGGTTTCACGAGGCTAGGTCCAGACTCCTGCAGCTGCACCT CGAATTCCCAR05C14-Light variable (SEQ ID NO: 28): TTGGTCCCCCCTCCGAACGTGTACGGCCAGTTGTTACTCTGTTGACAGAAATACATTCCAAAATCTTCAGTCTCCACACTGATGATACTGAGAGTGAAATCCGTCCCTGATCCACTGCCACTGAACCTGGAGGGGATCCCAGAGATGGACTGGGAAGCATACTTGATGAGAAGCCTTGGAGACTCATGTGATTTTTGTTGATACCAGTGTAGGTTGTTGCTAATACTTTGGCTGGCCCTGCAGGAAAGACTGACGCTATCTCCTGGAGTCACAGACAGGGTGTCTGGAGACTGAGTTAGCACAATATCACCTCTGGAGGCTGAAATCCAGAAAAGCAAAAAA

Example 24—Sequence Recovery for Amplification of Effector CellAntibodies

The following protocol and primers was used to recover antibodysequences from mouse, humans or rabbits.

Reagents for reverse transcription (RT) and polymerase chain reaction(PCR) are provided in Table 7.

TABLE 7 Reverse transcription (RT) reaction PCR reaction RNAseIn 2 U/μLPCR buffer 1× RNAse inhibitor Betaine 1M dNTPs 200 nM each First strand1× MgCl₂ 1 mM RT buffer DTT 2.5 mM Enzyme (KOD) 0.008 U/uL MgCl 2.9 mMUniversal 600 nM TS primer RT enzyme 1 U/μL Universal 500 nM (M-MLV) RTprimer Template 1.2 μM Long gene- 100 nM Switching specific primerprimers RT primers 40 nM each

The following RT-PCR thermal cycling protocol was followed:

RT: 3 min. at 72° C., 90 min. at 42° C., 10 min at 85° C.

Hot start: 2 min. at 95° C.; Denaturation: 15 sec. at 95° C.;Elongation: 15 sec. at 72° C.; Annealing: 15 sec. at the followingtemperatures: 72° C. 3 cycles; 70° C. 3 cycles; 68° C. 3 cycles; 66° C.3 cycles; 64° C. 3 cycles; 62° C. 6 cycles; 60° C. 20-30 cycles.

The primers used in the RT-PCR experiments are provided in Table 8, asfollows:

TABLE 8 List of Primers Name  Sequence SEQ ID NO: Template switching primer  GCAGTGGTATCAACGCAGAGTACG(r)G(r) SEQ ID NO: 29  G(r) Universal primer TS  GCAGTGGTATCAACGCAGAGTACG SEQ ID NO: 30  Universal primer RT  AGACAGTCCTCAGTGCCTCGTCAGCAG SEQ ID NO: 31  Human RT primer HsRT-01  gtcctgaggactg  SEQ ID NO: 32 Human RT primer HsRT-02  tgctctgtgacac  SEQ ID NO: 33 Human RT primer HsRT-03  ggtgtacaggtcc  SEQ ID NO: 34 Human RT primer HsRT-04  cagagagcgtgag  SEQ ID NO: 35 Human RT primer HsRT-05  tcatgtagtagctgtc  SEQ ID NO: 36 Human RT primer HsRT-06  ctcaggactgatgg  SEQ ID NO: 37 Human RT primer HsRT-07  gagtcctgagtactg  SEQ ID NO: 38 Human RT primer HsRT-08  gttgttgctttgtttg  SEQ ID NO: 39 Human RT primer HsRT-09  ttgttgctctgtttg  SEQ ID NO: 40 Long gene-specific primer  AGACAGTCCTCAGTGCCTCGTCAGCAGACCA SEQ ID NO: 41  HsN2U_01  GGCAGCCCAGGGC  Long gene-specific primer AGACAGTCCTCAGTGCCTCGTCAGCAGCAGT  SEQ ID NO: 42  HsN2U_02 GTGGCCTTGTTGGCTTGAAGCTCC  Long gene-specific primer AGACAGTCCTCAGTGCCTCGTCAGCAGAGCA  SEQ ID NO: 43  HsN2U_03 GGCACACAACAGAGGCAGTTCC  Long gene-specific primer AGACAGTCCTCAGTGCCTCGTCAGCAGGCCC  SEQ ID NO: 44  HsN2U_04 AGAGTCACGGAGGTGGCATTG  Long gene-specific primer AGACAGTCCTCAGTGCCTCGTCAGCAGGCAT  SEQ ID NO: 45  HsN2U_05 GCGACGACCACGTTCCCATCTTG  Long gene-specific primer AGACAGTCCTCAGTGCCTCGTCAGCAGGCAG  SEQ ID NO: 46  HsN2U_06 CCAACGGCCACGCTG  Long gene-specific primer AGACAGTCCTCAGTGCCTCGTCAGCAGATGC  SEQ ID NO: 47  HsN2U_07 CAGGACCACAGGGCTGTTATCCTTTG  Long gene-specific primer AGACAGTCCTCAGTGCCTCGTCAGCAGAGTG  SEQ ID NO: 48  HsN2U_08 TGGCCTTGTTGGCTTGGAGCTC  Long gene-specific primer AGACAGTCCTCAGTGCCTCGTCAGCAGACCA  SEQ ID NO: 49  HsN2U_09 CGTTCCCATCTGGCTGGGTG  RT mouse primer MmRT_01  acagtcactgagct SEQ ID NO: 50  RT mouse primer MmRT_02  ctttgacaaggcatc  SEQ ID NO: 51 RT mouse primer MmRT_03  ccacttgacattgatg  SEQ ID NO: 52 RT mouse primer MmRT_04  ctcttctccacagtg  SEQ ID NO: 53 Long gene-specific mouse  AGACAGTCCTCAGTGCCTCGTCAGCagactg SEQ ID NO: 54  primer Mmn2_01  caggagagctgggaaggtgtg Long gene-specific mouse AGACAGTCCTCAGTGCCTCGTCAGCaggaca  SEQ ID NO: 55 primer Mmn2_02  gctgggaaggtgtgcacac  Long gene-specific mouseAGACAGTCCTCAGTGCCTCGTCAGCtcaaga  SEQ ID NO: 56  primer Mmn2_03 agcacacgactgaggcacctcc  Long gene-specific mouseAGACAGTCCTCAGTGCCTCGTCAGCttgcct  SEQ ID NO: 57  primer Mmn2_04 tccaggccactgtcacacc  Long gene-specific mouseAGACAGTCCTCAGTGCCTCGTCAGCatccag SEQ ID NO: 58  primer Mmn2_05 atgtgtcactgcagccagggac  Long gene-specific mouseAGACAGTCCTCAGTGCCTCGTCAGCAccttc  SEQ ID NO: 59  primer Mmn2_06 cagtccactgtcaccacacctg  RT rabbit primer OcRT_01  TGAAGCTCTGGAC SEQ ID NO: 60  RT rabbit primer OcRT_02  CACACTCAGAGGG  SEQ ID NO: 61 RT rabbit primer OcRT_03  TTCCAGCTCACAC  SEQ ID NO: 62 RT rabbit primer OcRT_04  AGGAAGCTGCTG  SEQ ID NO: 63 RT rabbit primer OcRT_05  ACACTGCTCAGC  SEQ ID NO: 64 RT rabbit primer OcRT_06  TCACATTCAGAGGG  SEQ ID NO: 65 RT rabbit primer OcRT_07  GTCTTGTCCACTTTG  SEQ ID NO: 66 RT rabbit primer OcRT_08  CTCTGTTGCTGTTG  SEQ ID NO: 67 Long rabbit gene-specific AGACAGTCCTCAGTGCCTCGTCAGgatcagg SEQ ID NO: 68  primer Oc-PCR-IgHA1A7-12  cagccgacgacc Long rabbit gene-specific AGACAGTCCTCAGTGCCTCGTCAGgtgggaa SEQ ID NO: 69  primer Oc-PCR-IgKC1  gatgaggacagtaggtgc Long rabbit gene-specific AGACAGTCCTCAGTGCCTCGTCAGagatggt SEQ ID NO: 70  primer Oc-PCR-IgKC1KC2  gggaagaggaggacag Long rabbit gene-specific AGACAGTCCTCAGTGCCTCGTCAGccttgtt SEQ ID NO: 71  primer Oc-PCR-IgLC4L5L6  gtccttgagttcctcagagg Long rabbit gene-specific AGACAGTCCTCAGTGCCTCGTCAGcggatca SEQ ID NO: 72  primer Oc-PCR-IgA2A6  ggcagccgatgac Long rabbit gene-specific AGACAGTCCTCAGTGCCTCGTCAGcaggtca SEQ ID NO: 73  primer Oc-PCR-IgA4A5  gcgggaagatgatcg Long rabbit gene-specific AGACAGTCCTCAGTGCCTCGTCAGcactgat SEQ ID NO: 74  primer Oc-PCR-IgLC1C2C3  cagacacaccagggtgg Long rabbit gene-specific AGACAGTCCTCAGTGCCTCGTCAGcaccgtg SEQ ID NO: 75  primer Oc-PCR-IgG  gagctgggtgtg Long rabbit gene-specific AGACAGTCCTCAGTGCCTCGTCAGgatcagg SEQ ID NO: 76  primer Oc-PCR-IgA3A5  cagccggcgatc Long rabbit gene-specific AGACAGTCCTCAGTGCCTCGTCAGggagacg SEQ ID NO: 77  primer Oc-PCR-IgM  agcgggtacagagttg Long rabbit gene-specific AGACAGTCCTCAGTGCCTCGTCAGictgcag SEQ ID NO: 78  primer Oc-PCR-IgE  caggaggccaag 

FIG. 103A shows a micrograph of PCR amplicons produced as describedabove and testing a gradient of RT temperatures ranging from 60° C. to40° C. Template was 200 pg (˜ten cell equivalents) of RNA purified fromhybridoma cells (D1.3). Far right lane shows optimized condition usingKOD polymerase. HV and LV amplicons are expected within 450 to 550 bpwhere strong bands are observed; this region also includes somenon-specific products. Referring to FIG. 103B, band from 400 to 600 bpwas extracted and Sanger sequenced using a primer that annealed to aconstant region on the heavy chain. The trace showed the junction madeby MMLV between template switching oligo and cDNA, joined by CCC that isadded by MMLV during cDNA synthesis. The sequence was aligned andconfirmed to match the variable region sequence of the heavy chain ofD1.3.

Example 25—NGS Sequencing of Single Cells Recovered from Device

A different approach to retrieve antibody sequences combinestemplate-switching and next-generation sequencing. Referring to FIG. 59,single cells are deposited into microfuge tubes and cDNA is generatedfrom multiplexed gene-specific primers targeting the constant region ofheavy and light chains. Template-switching activity of MMLV enzyme isused to append the reverse complement of a template-switching oligo ontothe 3′ end of the resulting cDNA. Semi-nested PCR, using multiplexedprimers that anneal to the constant region of heavy and light chain anda universal primer complementary to the copied template switchingoligonucleotide, is used to amplify cDNA and introduce indexingsequences that are specific to each single cell amplicon. Amplicons arethen pooled and sequenced.

Another approach to recover sequences from heterogeneous populations ofcells couples microfluidic single cell antibody analysis with Ig-Seq(FIG. 104A) Following immunization, ASCs are collected from the animal;a fraction are analyzed on microfluidic devices while the remaining areused for construction of a bulk amplicon library for Ig-Seq. From themicrofluidic device, a total of 96 indexed single cell (SC) librariesand 96 indexed low diversity (LD) libraries are pooled for sequencing onMiSeq. Analysis of the bulk library is used to determine HV and LVclonotypes present in the immune response, shown as clusters in FIG.104B. SC libraries provide paired chain HV and LV sequences of mAbs frommost abundant clonotypes that are confirmed to be antigen specific. LDlibraries provide additional identification of HV and LV sequences thatare antigen specific or that are not antigen specific. LD libraries arealso used to infer chain pairing by analysis of co-occurrence of HV andLV sequences across LD libraries, illustrated in FIG. 104C. Informationon binding status and chain pairing for specific sequences allowsinterpretation of the bulk sample by assignment of binding status,represented as stars (antigen-specific) and crosses (non-specific) inFIG. 104B, and clonotype pairing.

Example 26—Multiplexed Bead Assay Using Optically Encoded Beads

The multiplexed bead based assay in this example allows the measurementof several different antigen specific antibodies in the same chamberfrom antibody secreting cells. Fluorescence intensity coded beads (e.g.,Starfire Red™ dye beads, Bangs laboratories) can be used to trackdifferent antigen specific antibodies by labeling each subset of beadswith a different antigen using a traditional sulfo-NHS(N-hydroxysulfosuccinimide)/1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride (EDC) protein coupling strategy (Pierce). Starfire Red™(excitation from UV to red, emission max at 675 nm) and antigen specificfluorescence signal can then be measured and quantified.

Starfire Red™ dye beads (5.5 μm diameter) containing 3 subsets offluorescently coded beads were coated with different 2012-2013 seasonalflu specific antigens (FIG. 105A). Beads (from the least intense to themost intense Starfire Red™ intensity respectively) were coupled withH1N1 (A/California/7/2009 (H1N1) pdm09-like strain), H3N2(A/Victoria/361/2011 (H3N2)-like strain) and B strain(B/Wisconsin/1/2010-like strain) antigens (Protein Science Corp.).

Beads were mixed in a 1:1:1 ratio and were injected in a microfluidicdevice (FIG. 105B). Starfire Red™ dye fluorescent measurement revealsthe positions of each particular subset of beads and allows furthersubsequent tracking of antigen specific antibody binding (FIG. 105C).Rabbit anti-H1N1 specific antibody (Sino Biological) dissolved in mediawas then flown in the device at a concentration of 400 pg/mL andincubated for 45 minutes. Anti-rabbit IgG labeled with Dylight 488(Jackson Immunoresearch) was then sent in the device and fluorescentimaging was performed. Positive signal is clearly visible on the H1N1coated beads (least bright Starfire Red™ beads represented by arrows)(FIG. 105D) and no unspecific binding is detected on the beads labeledwith H3N2 and B strain specific antigens (FIG. 105E).

Example 27—Microfluidic Apoptosis Effector Assay

A functional extracellular effect assay was implemented to findantibodies and effector cells that produce such antibodies, thatneutralize TNF-α or that block its receptor. To quantify the effect ofTNF-α on target L929 cells (readout cells), the readout cells werestained with DiOC₁₈ and incubated in the presence of 1 μg/mL ofactinomycin-D and different concentrations of TNF-α in multiwell plates.After 24 hours, cells were counterstained with propidium iodide (PI) andcell viability was measured by counting the fraction of PI-stained cellsfrom DiOC18-stained cells under a microscope. The dose response of TNF-αon L929 cells is shown in FIG. 106A.

In a second experiment, L929 cells were stained with CFSE, loaded in amicrofluidic device and incubated in the presence of 10 μg/mL ofactinomycin-D and 10 ng/mL of TNF-α and tracked by time-lapse imaging.Cells underwent rapid apoptosis, which was confirmed by PI labeling at24 hours (FIG. 106B).

Example 28—Microfluidic Cell Signaling Effector Assay

A TNFα functional assay based on nuclear or cytoplasmic fluorescencelocalization was developed for the function-based selection ofantibodies against TNFα. The readout cell line used was previouslydescribed in Tay et al. (2010). Nature 466, pp. 267-71, incorporated byreference herein in its entirety. Upon TNFα-induced activation, afluorescently-labeled NF-κB transcription factor subunit is transportedfrom the cytoplasm to the nucleus. FIG. 107A shows time-lapsefluorescence images for the TNFα functional assay. In the absence ofTNFα ligand, fluorescence localization is cytoplasmic (FIG. 107A, upperpanels). Upon activation by TNFα ligand (10 ng/mL), a change influorescence from cytoplasmic to nuclear is observed (FIG. 107A, middlepanels). In the presence of cell supernatant containing an antibody thatneutralizes TNFα ligand in addition to TNFα ligand (10 ng/mL), thefluorescence localization remains cytoplasmic (FIG. 107A, lower panels),indicating that TNFα ligand has been effectively neutralized by theantibody thus preventing NFκB signaling. The fraction of activated cellsfor each condition is shown in FIG. 107B.

Example 29—Microfluidic Proliferation and Autophagy Effector Cell Assay

A human breast cancer cell line (SKBR3) overexpressing HER2 andengineered with an LC3-GFP autophagy reporter was loaded in amicrofluidic device and cultured for 3 days to determine the feasibilityof using this cell line as readout cells. (FIG. 108). Individual SKBR3cell populations were provided to individual microfluidic chambers.Time-lapse imaging in bright field and fluorescence showed an increasein the number of cells over time (FIG. 108). The results indicated thatthe SKBR3 cells are suitable to use as readout cells in extracellulareffect assays that measure an effector cell's ability/propensity toblock the proliferation of SKBR3 cells. Using the SKBR3cell line it isalso possible to screen for antibodies that modulate autophagy.

Example 30—Detection and Recovery of Antigen Specific T Cells

Referring to FIG. 109, peripheral blood mononuclear cells (PBMCs) from ahuman patient were loaded in a microfluidic device and stimulated with apool of viral antigens derived from Cytomegalovirus, Epstein-Barr Virusand Influenza Virus (CEF) peptides at 10 μg/mL overnight. FIG. 109Ashows a bright field image of a microfluidic chamber loaded with PBMCsand interferon-γ (IFNγ) capture beads. Upon stimulation with CEFpeptides, IFNγ secreted by activated antigen-specific T cells wascaptured on functionalized beads coated with anti-IFNγ antibody anddetected with a fluorescently labeled secondary antibody. FIG. 109B is afluorescent image of the positive chamber from FIG. 109A containing anactivated antigen-specific T cell. Activated cells were expanded with100 U/mL of interleukin-2 (IL-2) for 5 days (FIG. 109C) and weresubsequently recovered. The frequency of CEF-specific T cells inperipheral blood mononuclear cells from the same patient was measured byELISPOT and the microfluidic bead assay for comparison. The sensitivityof the microfluidic assay allowed for the detection of higherfrequencies of antigen-specific T cells than ELISPOT (FIG. 109D).

Example 31—BAF3 PDGFRα Assay for Signaling

A cell survival assay was generated for the function-based selection ofantibodies against PDGFRα. A BaF3 suspension cell line, dependent on thecytokine IL-3 for survival and growth, was electroporated with humanPDGFRα driven by a CMV promoter (Origene) and stably expressing cloneswere generated. The PDGFRα overexpressed in Ba/F3 cells substitutes forthe requirement of IL-3 signaling; in the absence of IL-3 BaF3 cellsarrest and die, but in the presence of PDGF ligand PDGFRα signalingrescues these cells, giving a cell survival and mitogenic response thatis easily detected by microscopy, including proliferation, morphologicalchanges, and increased motility/chemotaxis. As a fluorescence readout,BaF3 cells were also stably expressing histone 2B fused to a yellowfluorescent protein (YFP) to label cell nuclei. FIG. 110 showsvalidation of cell survival PDGFRα functional assay, showing a BaF3clone expressing PDGFRα and histone 2B-YFP in the presence of no ligandor PDGF-AA 25 ng/mL for T=48 hours. In the absence of ligand, BaF3 cellsundergo apoptosis and loss of YFP fluorescence (likely a result ofprotein degradation upon apoptosis) (FIG. 110A). In the presence ofPDGF-AA ligand, cell survival and growth are rescued as shown by YFPfluorescence readout (FIG. 110B).

To test whether antibody-secreting cells could be kept viable for thelength of such an assay, mouse splenocytes were enriched for plasmacells and co-incubated with BaF3 cells overexpressing PDFGRα in amicrofluidic device. Bright field and fluorescent images were taken atthe beginning of the experiment to distinguish the effector cellpopulation from the readout cell population (containing H2B-YFP). Cellswere cultured in the presence of IL-3 and tracked by time-lapse imagingfor 48 hours, at which point anti-mouse antibody capture beads wereintroduced in the device for a 2-hour incubation. Chambers containingantibody-secreting cells were identified using a Dylight-594 labeledantibody. FIG. 110C shows an example of a microfluidic chambercontaining 2 splenocytes (black arrows) and 2 readout cells (whitearrows) at the beginning of the experiment. Readout cells proliferatedto 22 cells while the effector cell population kept secreting antibodiesafter 48 hours of culture.

Example 32—GPCR Response to Ligand Using DiscoveRx Cells

In the PathHunter® β-Arrestin system from DiscoveRx (www.discoverx.com),a small peptide is fused to the intracellular sequence of a GPCR targetof interest, and a complementing peptide fragment is fused to anotherintracellular protein (β-arrestin). After binding to its specificligand, the GPCR recruits 0-arrestin, forcing the complementation of thetwo peptides to produce a functional β-galactosidase enzyme. In theconventional non-microfluidic assay, the enzyme activity, and thus theamount of ligand bound to the GPCR, is detected with a single additionof a reagent cocktail to lyse the cells and produce a chemiluminescentbefore being analyzed using a traditional plate reader.

This assay was modified to adapt it to the microfluidic format describedherein by using a fluorescent based substrate.5-Dodecanoylaminofluorescein Galactopyranoside (C₁₂FDG) is a chemicallymodified and non-fluorescent β-galactosidase substrate that becomesfluorescent after enzyme cleavage. It also includes a lipophilic tailthat allows the substrate to diffuse inside the cell membrane and alsopromotes the retention inside the cells after cleavage.

The CCR4/CCL22 GPCR-agonist pair (PathHunter® eXpress CCR4 CHO-K1β-Arrestin GPCR Assay) was used as an example to adapt the assay withthe C12FDG substrate. Cells were incubated with various concentrationsof substrate and ligand before being imaged using fluorescence.

The adapted assay was first tested in multiwell plates. Cells wereincubated in media for 90 minutes with various concentrations of C₁₂FDGsubstrate. The substrate diffused in the cells and remained nonfluorescent until it was cleaved. Then, the ligand/agonist CCL22 wasadded at different concentrations and incubated for 60 minutes for thecomplementation to occur inside the cells, leading to the formation ofβ-galactosidase and cleavage of the substrate, producing a fluorescentproduct inside the cells. Fluorescence-based microscopy was then used toimage each well corresponding to a particular condition (FIG. 111).

Cells were then loaded in a microfluidic device and incubated with 33 μMC₁₂FDG substrate in cell culture media. The substrate and the cells wereincubated for 90 minutes in order to allow diffusion and accumulation ofthe non-fluorescent substrate inside the cells. Cells were washed withmedia for 10 minutes at the end of the incubation. CCL22 agonist/ligandwas then loaded at 4 different concentrations in 4 different sub-arraysof the device (sub-array 1: 0.01 nM, sub-array 2: 1 nM, sub-array 3: 100nM, sub array 4: no agonist). Fluorescence-based microscopy was thenused to image each chamber corresponding to a particular condition (FIG.112A). Image analysis was performed for each chamber and averageintensity was measured in the chambers. Background subtraction wasperformed and the results were plotted in FIG. 112B.

All, documents, patents, patent applications, publications, productdescriptions, and protocols which are cited throughout this applicationare incorporated herein by reference in their entireties for allpurposes.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Modifications and variationof the above-described embodiments of the invention are possible withoutdeparting from the invention, as appreciated by those skilled in the artin light of the above teachings. It is therefore understood that, withinthe scope of the claims and their equivalents, the invention may bepracticed otherwise than as specifically described.

1-218. (canceled)
 219. A method of identifying an antibody secretingcell (ASC) that secretes a virus neutralizing antibody, comprising:retaining in a plurality of microreactors a plurality of cellpopulations, wherein individual cell populations of the pluralitycomprise one or more ASCs, wherein the contents of individualmicroreactors of the plurality of microreactors each comprise a readoutparticle population comprising one or more readout particles, andwherein the individual cell populations are retained in the individualmicroreactors; introducing a plurality of accessory particle populationsinto the individual microreactors, wherein individual accessory particlepopulations of the plurality comprise a plurality of virus particles andthe individual accessory particle populations are retained in individualmicroreactors; incubating the individual cell populations, the readoutparticle populations and the accessory particle populations within theindividual microreactors to provide secreted antibodies; assaying theindividual microreactors for the presence of a virus neutralizingantibody, determining, based on the results of the assaying step,whether one or more of the cell populations comprises an ASC thatsecretes a virus neutralizing antibody.
 220. The method of claim 219,wherein the readout particle population comprises a homogeneouspopulation of readout particles.
 221. The method of claim 219, whereinthe readout particle population comprises a heterogeneous population ofreadout particles.
 222. The method of claim 220, wherein the homogeneouspopulation of readout particles comprises a homogeneous population ofreadout cells.
 223. The method of claim 221, wherein the heterogeneouspopulation of readout particles comprises a heterogeneous population ofreadout cells.
 224. The method of claim 219, wherein the readoutparticle population or subpopulation thereof is immobilized on a surfaceof the individual microreactors.
 225. The method of claim 219, furthercomprising maintaining the individual cell populations in substantiallya single plane.
 226. The method of claim 219, further comprisingmaintaining the readout particle populations in substantially a singleplane.
 227. The method of claim 219, wherein the plurality of accessoryparticles further comprise a fluorescent substrate, fluorophore, asecondary antibody, or a combination thereof.
 228. The method of claim219, wherein the individual accessory particle populations of theplurality are heterogeneous accessory particle populations.
 229. Themethod of claim 219, wherein the plurality of virus particles isengineered to include a fluorescent protein expressed by a readout cellfollowing virus infection of the readout cell.
 230. The method of claim219, wherein assaying the individual microreactors comprises assayingthe morphology of the readout particle populations.
 231. The method ofclaim 219, wherein assaying the individual microreactors comprisesassaying binding of the secreted antibodies to the virus particles. 232.The method of claim 219, wherein assaying the individual microreactorscomprises assaying the expression of fluorescent proteins within thereadout particle populations that are upregulated during viralinfection.
 233. The method of claim 219, wherein assaying the individualmicroreactors comprises assaying the death of the readout particlepopulations.
 234. The method of claim 219, further comprisingsubstantially isolating the individual microreactors from theirsurrounding environments.
 235. The method of claim 219, wherein if acell population comprises an ASC that secretes a virus neutralizingantibody, the method further comprises recovering the cell populationcomprising the ASC that secretes the virus neutralizing antibody or aportion thereof to obtain a recovered cell population.
 236. The methodof claim 230, wherein the recovering step comprises positioning the openend of a microcapillary in a microreactor comprising the cell populationcomprising the ASC that secretes the virus neutralizing antibody andaspirating the microreactor's contents or a portion thereof to obtain arecovered aspirated cell population.
 237. The method of claim 236,wherein the microcapillary is mounted on a robotic micromanipulationsystem on a microscope or the microcapillary is controlled robotically.238. The method of claim 235, further comprising, retaining a pluralityof cell subpopulations originating from the recovered cell population ina plurality of vessels, wherein each cell subpopulation is present in anindividual vessel, lysing the individual cell subpopulations to providelysed cell subpopulations, and amplifying one or more nucleic acidswithin each of the lysed cell populations.